Immunelektrophoresis

 

The principle of immunoelectrophoresis is the formation of precipitate lines at the equivalence point of the antigen and its corresponding antibody. In this method it is important that the ratio between the quantities of antigen and antibody be correct (antibody titer). When the antibody is in excess, statistically at most one antigen binds to each antibody while when the antigen is in excess at most one antibody binds to each antigen. Yet at a specific antigen/antibody ratio (equivalence point) huge macromolecules are formed. They consist of an antigenantibodyantigenantibody... sequence and are immobilized in the gel matrix as an immunoprecipitate. The white precipitate lines are visible

Besides immunofixing and immunoprinting, immunoblotting also exists for protein identification: immobilizing membranes, for example nitrocellulose, are used on the surface of which the proteins are adsorbed, see "blotting' in the gel and can be revealed with protein stains. The method is specific and the sensitivity very high because distinct zones are formed. Immunoelectrophoresis can be divided into three principles:

 

A. Counter immunoelectrophoresis according to Estela and Heinrichs : in an agarose gel exhibiting high electroosmosis, the buffer is set at a pH about 8.6 so that the antibody does not carry any net charge. The sample and the antibody are placed in their respective wells and move towards each other: the charged antigens migrate electrophoretically and the antibodies are carried by the electroosmotic flow.

 

B. Zone electrophoresis immunodiffusion according to Graber and Williams : first a zone electrophoresis is run in an agarose gel, followed by the diffusion of the antigen fraction towards the antibody which is pipetted into troughs cut in the side parallel to the electrophoretic run.

 

C. The "rocket" technique according to Laurell : antigens migrate in an agarose gel which contains a definite concentration of antibody. As in method A the antibodies are not charged because of the choice of the buffer. As the sample migrates one antibody will bind to one antigen until the ratio of concentrations corresponds to the equivalence point of the immunocomplex.

The result is that rocket shaped precipitin lines are formed, the enclosed areas are proportional to the concentration of antigen

A series of modifications to this technique exist. including twodimensional ones.

 

 Affinity electrophoresis

 

This is a method related to immunoelectrophoresis which is based on the interactions between various macromolecules for example lectinglycoprotein, enzymesubstrate and enzymeinhibitor complexes

All the known immunoelectrophoretic techniques are used here. For example, specific binding lectin collected worldwide from plant seeds are examined with line affinity electrophoresis. In this way carbohydrate changes in glycoproteins during different biological processes can be identified.