Immunoelectrophoresis

Agar Gel and Immunoelectrophoresis

Immunoelectrophoresis is a widely used technique which consists of a primary electrophoretic separation of proteins in an electric field, followed by immunoprecipitation. The primary electrophoresis, is utilized to separate the serum proteins in an agar gel. These serum proteins are then allowed to diffuse against antibodies, antiserum, added to the trough found within the middle of the gel, which eventually leads to a patter of precipitation arcs at points of equivalence. These arcs represent the major, highly abundant constituents of the serum proteins. Such a technique proves useful when multiple antigen-antibody complexes must be resolved. It can resolve similar diffusion property antigens, which differ in their electrophoretic mobility. This method also is far faster and sensitive to the previous mentioned procedures, because it initially separates the antigens based on their electrophoretic ability in a charged field, followed by antibody specificity separation. Unreacted proteins are washed off the gel, which is later stained, in order to visualize the precipitin bands.

A simple experiment you can conduct is agar gel electrophoresis of bovine serum and BSA, with immunoelectrophoresis with anti-BSA was perormed. The first gel is of bovine serum and BSA. The constituents within each sample were electrophoresized through the gel and separated based on their overall charge (electrophoretic mobility). Therefore, the negatively charged constituents migrated towards the cathode. This separated the serum proteins into defined regions, and apart, distinguishable from one another. BSA, an albumin on the bottom well, migrated towards anode. Addition of the trough between the electrophoresed proteins and addition of polyclonal anti-BSA allowed for immunodiffusion. The precipitin reactions arcs formed more densely at the location of the migrated BSA in the bovine serum, and less intensely throughout the rest of the proteins.

The BSA and anti-BSA formed a similar dense arc at the location of BSA, in the BSA solution. The presence of precipitin reaction throughout the bovine serum indicates that the polyclonal anti-BSA is cross-reacting with other antigens in the bovine serum. In addition, antibodies found within the bovine serum may cross-react to the serum, and also there can be poor protein resolution during electrophoresis. Moreover, there can be very poor destaining, due to no destainer being left, as the gel can be fairly dark, obscuring a good interpretation of the results. TO remove any cross-reactivity, you could add pre-immunized serum from the animal which you got the anti-BSA from and add it to the bovine serum, electrophoresed. Overall, with more available resources, and more gels being run, better results can be obtained.

Immunoelectrophoresis

Immunoelectrophoresis is a widely used tool for analyzing immunoglobulin abnormalities, such as macroglobulinemia. Immunoglobulins are produced by bone marrow derived plasma cells, which if they become tumourgenic, myeloma cells, then abnormally large quantities of immunoglobulins are produced and secreted into the serum. Appearance of these myelomas, decreases the amount, often, of other serum proteins. The utilization of antisera against the various heavy and light chains of the immunoglobulin molecules allows for the detection of abnormal elevated and decreased levels of patient immunoglobulin molecules through a patient versus normal serum immunoelectrophoresis. The appearance of different precipitin bands in the patient versus normal serum, can be indicative of a pathology, such as myeloma. The patient with the abnormal serum usually contains characteristic curves and expression levels that have a different length, thickness, positioning (distance from well), and intensity of the precipitin arc band compared to normal serum.

References:

1. Janeway, C.A., Travers, P., Walport, M., and Capra, J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA.

2. Delves, P., and Roitt, I. 1999. Encyclopedia of Immunology. Academic Press Inc., 2nd ed., San Diego, USA.

3. Cruse, J. and Lewis, R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA.

4. Bryant, N. 1986. Laboratory Immunology and Serology. B. Venable, W.B. Saunders Company, 1st ed., Philadelphia, USA.