IMMUNOELECTROPHORESIS

Immunoelectrophoresis is a technique that separates proteins on the basis of both their net charge (and so their movement in an electric field) and on the response of the immune system to the proteins. The technique is widely used in both clinical and research laboratories as a diagnostic tool to probe the protein composition of serum.

Petr Nikolaevich Grabar, a French immunologist, devised the technique in the 1950s. In essence, immunoelectrophoresis separates the various proteins in a sample in an electric field and then probes the separated proteins using the desired antiserum.

The most widely used version of the technique employs an apparatus, which consists basically of a microscope slide-sized plate. The plate is the support for a gel that is poured over top and allowed to congeal. The construction of the gel can vary, depending on the separation to be performed. Agar, such as that used in microbiological growth media, and another material called agarose can be used. Another popular choice is a linked network of a chemical known as acrylamide. The linked up acrylamide chains form what is designated as polyacrylamide.

The different types of gel networks can be most productively envisioned as a three-dimensional overlay of the crossed linked chains. The effect is to produce snaking tunnels through the matrix of various diameters. These diameters, which are also referred to as pore sizes, can be changed to a certain extent by varying the concentrations of some of the ingredients of the gel suspension. Depending on the size and the shape of the protein, movement through this matrix will be relatively slow or fast. As well, depending on the net charge a protein molecule has, the protein will migrate towards the positively charged electrode or the negatively charged electrode when the electric current is passed through the gel matrix. Thus, the various species of protein will separate from each other along the length of the gel.

In some configurations of the immunoelectrophoretic set-up, the samples that contain the proteins to be analyzed are added to holes on either side of the gel plate. For example, one sample could contain serum from a health individual and another sample could contain serum from someone with an infection. The middle portion of the plate contains a trough, into which a single purified species of antibody or known mixture of antibodies is added. The antibody molecules diffuse outward from the trough solution into the gel. Where an antibody encounters a corresponding antigen, a reaction causes the formation of a visual precipitate. Typically, the precipitation occurs in arc around the antigen-containing sample. In the example, the pattern of precipitation can reveal antigenic differences between the normal serum and the serum from a infected person.

This type of immunoelectrophoresis provides a qualitative ("yes or no") answer with respect to the presence or absence of proteins, and can be semi-quantitative. The shape of the arc of precipitation is also important. An irregularly shaped arc can be indicative of an abnormal protein or the presence of more than one antigenically similar protein.

Immunoelectrophoresis can also be used to detect a particular antigenic site following the transfer of the proteins from a gel to a special support, such as nitrocellulose. Addition of the antibody followed by a chemical to which bound antibody reacts produces a darkening on the support wherever antibody has bound to antigen. One version of this technique is termed Western Blotting. An advantage of this technique is that, by running two gels and using just one gel for the transfer of proteins to the nitrocellulose, the immune detection of a protein can be performed without affecting the protein residing in the other gel.

Another application of immunoelectrophoresis is known as capillary immunoelectrophoresis. In this application, a sample can be simultaneously drawn up into many capillary tubes. The very small diameter of the tubes means that little sample is required to fill a tube. Thus, a sample can be subdivided into very many sub volumes. Each volume can be tested against a different antibody preparation. Often, the reaction between antigen and antibody can be followed by the use of compounds that fluoresces when exposed to laser light of a specific wavelength. Capillary immunoelectrophoresis is proving to be useful in the study of Bovine Spongiform Encephalopathy in cattle, where sample sizes can be very small.

In the clinical laboratory setting, immunoelectophoresis is used to examine alterations in the content of serum, especially changes concerned with immunoglobulins. Change in the immunoglobulin profile can be the result of immunodeficiencies, chronic bacterial or viral infections, and infections of a fetus. The immunoglobulin most commonly assayed for are IgM, IgG, and IgA. Some of the fluids that can be examined using immunoelectrophoresis include urine, cerebrospinal fluid and serum. When concerned with immunoglobulins, the technique can also be called gamma globulin electrophoresis or immunoglobulin electrophoresis.