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Peter V. Ostoich Serum Protein Electrophoresis Definition: A serum protein electrophoresis (SPE, SPEP or EPG) is a classic method used for the separation of proteins from blood serum (the fluid portion that remains after the blood has clot). In SPE the proteins are separated according to their size and electric charge. SPE can refer to three main methods of separation: a) Separation in a glass or plastic migration trough (oldest method) b) Separation of serum proteins on agarose or polyacrylamide gels c) Separation on special cellulose acetate sheets, filter paper or agarose gelcovered paper (Fig. 1) subjected to charge applied at both ends (standardized clinical test) The proteins can be visualized by general stains (Coomassie Brilliant Blue, Ponceau S, silver staining), immunoprecipitation traces formed after antibody incubation or immunofluorescence. Variations: Immunoelectrophoresis (IEP) is a sub-class of SPE, in which specific antisera are used to immunoprecipitate target proteins in the sample (Fig. 2); IEP is used most often for detection of increased IgG/IgM or appearance of monoclonal antibodies associated with myelomas. Fig. 1: Agarose-film membrane for SPE, Cypress Diagnostics Fig. 2: Immunoelectrophoresis for diagnosis of myeloma (arrow denotes anomalous precipitation pattern) Applications: Serum electrophoresis is routinely used as a medical diagnostic tool. When a serum protein is needed in large amounts (i.e. bovine serum albumin), the classic method of obtaining it is by SPE. Human _-globulins are purified from sera by SPE and are subsequently administered to patients who have developed a life-threatening infection (meningitis) or are at a high risk of secondary infection (burn victims). In immunology SPE is used for purification and/or quantification of any protein component of sera. 1 The SPE fractions: Originally the serum proteins were defined by their electrophoretic properties (Fig. 3); many proteins were named in accordance to the fraction from which they were isolated. There are five main "clusters": Albumin, _1, _2, _ and _. The _1, _2, _ and _ proteins are collectively known as globulins. Albumin is a negatively charged protein that serves to maintain the balance of body fluids. Bisalbuminemia (two bands) is observed in heterozygous individuals possessing two different albumin genes. Albumin deficiency can indicate liver disease or pancreatitis. The _1-zone is mainly composed of the _1-antitrypsin (the absence of which causes lung emphysema and liver destruction by trypsin) and orosmucoid proteins. Fig. 3 : Agarose SPE, Coomassie BB staining The _2-zone comprises _2macroglobulin and hepatoglobulin. During acute-phase response hepatoglobulin levels increase as hepatoglobulin is used to bind and target for phagocytosis free hemoglobin. The _-zone consists mostly of transferrin (a protein, which scavenges iron from the plasma, denying it to pathogens) and complement protein 3 (C3). _-2-microglobulin (secreted _-chain of MHC class 1) can also be found in the _-zone. Normally, the vast majority of the proteins present in the _-zone are immunoglobulins. IgA migrates to the intermediate region between the _ and _ regions, high levels of IgA occur in lung inflammation, rheumatoid arthritis or liver cirrhosis. Faint bands in the _zone indicate specific response to antigenic challenge; distinct narrow bands indicate the presence of high titers of monoclonal antibodies. Very prominent bands might indicate myelomas, lymphomas or leukemia. A substantial increase of total IgG (hypergammaglobulinemia) can occur in rheumatoid arthritis and chronic viral infection. References: 1. Reeves et al., Suspected Myeloma: Investigation of Paraproteinemia, Hunter Area Pathology Resources, 2001 2. Keyser, J. and Watkins, G. Estimation of Serum Proteins by Electrophoresis on Cellulose Acetate, Clinical Chemistry Vol.12 No.12, 1972 3. University of Virgina online tutorials in Basic Hematology, 2005 < http://www.med-ed.virginia.edu/courses/path/innes/> 2