Download 1 Peter V. Ostoich Serum Protein Electrophoresis Definition: A

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.
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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/>
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