Principle
Immunofixation electrophoresis (IFE) is a two-stage procedure using agarose gel high-resolution electrophoresis in the first stage and immunoprecipitation in the second. Proteins are first separated by electrophoresis. Then, in the second stage, soluble antigen in the gel reacts with specific antibody, and the resultant antigen–antibody complexes become insoluble (as long as the antibody is in slight excess or near equivalency) and precipitate. The precipitation rate depends on the proportions of the reactants, temperature, salt concentration, and the solution’s pH. The unreacted proteins are removed by a washing step, and the antigen– antibody complex is visualized by staining. The bands in the individual lanes are compared with the precipitin bands obtained with a normal control serum.
Sample Preparation
Fresh serum or urine is the specimen of choice. Evaporation of uncovered specimens may cause inaccurate results, so specimens must be handled carefully. Plasma should not be used, because the fibrinogen may adhere to the gel matrix, resulting in a band in all patterns across the gel. If storage is necessary, samples may be kept covered at 2°C to 8°C for up to 72 hours.