Immunoelectrophoresis

Immunoelectrophoresis is a general name for a number of biochemical methods for separation and characterization of proteins 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. Immunoelectrophoresis is a general term describing many combinations of the principles of electrophoresis and reaction of antibodies, also known as immunodiffusion. Agarose as 1% gel slabs of about 1 mm thickness buffered at high pH (around 8.6) is traditionally preferred for electrophoresis and 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. Electrophoresis equipment with a horizontal cooling plate was normally recommended for the electrophoresis. Immunoprecipitates are visible 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.