Normal Serum Proteins
Serum is composed of hundreds of different proteins and the total protein concentration and the concentration of several specific proteins is of clinical value. Serum proteins are resolved into 5-6 bands by electrophoresis, as commonly performed in the typical clinical laboratory. A typical electrophoretic separation of normal serum proteins may be seen by clicking on Serum Protein Electrophoresis in the table of Contents to the left. Return to here by clicking on Normal Serum Proteins.

The normal range for total serum protein is 6-8 g/dl. Somewhat more than half of total serum protein (3.5-5 g/dl) is accounted for by albumin, a homogeneous protein synthesized by the liver. The remaining total serum globulin fraction (2-3.5 g/dl) is comprised mainly by various transport proteins and acute phase reactant proteins (in the alpha and beta bands) synthesized by the liver, and by immunoglobulins in the gamma band synthesized by plasma cells.

Total serum protein and albumin are determined directly in a routine manner; total serum globulin is determined by the difference:
     total protein + Biuret reagent  ------>  purple colored
                            (alkaline Cu++)              Cu-peptide bond
     albumin + BromCresyl Green  ------>  intense green colored
                                                                       Alb-BCG complex

     serum globulin  =  total protein  -  albumin

Clinically important serum proteins of lower concentration are measured 
by specific techniques:

1.  lipoproteins              --   lipid staining of electropheretic strips, 
                                   serum lipid determinations,  
                                   determination of apoproteins

2.  thyroxin binding          --   radiolabeled tri-iodothyronine (T3) 
     globulin (TBG)                binding capacity

3.  transferin                --   iron binding capacity 
                                   or immunochemical determination
4.  alpha-1-antitrypsin            |
5.  IgG, IgA or IgM                | 
                                    >  immunochemical determination
6.  C3 or C4 components            | 
    of complement                  | 
7.  C-reactive protein (CRP)      _|                               

Pathologic Serum Protein Concentrations
Somewhat more than half of total serum globulin is accounted for by immunoglobulin in the gamma band. Noticeable alterations in total serum protein are almost always due to alterations in immunoglobulins and/or albumin; because concentrations of individual proteins in the alpha and beta bands are relatively low, changes in their concentrations rarely affect total protein or total serum globulin values. Albumin is elevated only in dehydration, but is decreased in a number of pathologic conditions. Increased total protein or total serum globulin is almost always due to increased immunoglobulins. Many of the individual alpha and beta globulins are of clinical significance and are determined by specific quantitative techniques.

Serum protein electrophoresis

Serum protein electrophoresis may be requested when total serum protein, albumin and/or total serum globulins are found to be abnormal. Electrophoresis is conducted by applying specimen at the origin, indicated by the dashed line in the figure to the right, on a cellulose acetate or agarose electrophoresis plate. A high voltage causes proteins to migrate at a rate determined by their net electrostatic charge and their size. Proteins are visualized by using a dye which stains all proteins in an equivalent manner. Protein staining with a red dye is illustrated in the example.

Certain other particular proteins may be visualized by using appropriate stains; for example lipoproteins are observed by using a fat stain, and isoenzymes are demonstrated using a substrate ---> product stain. A recorder tracing of the stained electrophoretic strip is obtained by scanning with a densitometer, which also provides quantification of the individual bands.

Pathologic Serum Protein Electrophoresis Patterns
A number of pathologic conditions are associated with a characteristic serum protein electrophoresis pattern, some of which are illustrated below.

Hepatic Cirrhosis --

When liver function is sufficiently diminished, protein synthesizing capacity is compromised and concentrations of albumin and proteins in the alpha and beta bands are decreased. An additional common finding is beta-gamma bridging due to increased IgA.

The Nephrotic Syndrome --

Renal disease involving the glomeruli is always associated with increased urinare protein loss. When protein loss is greater than 3-4 g/day, the protein synthesizing capacity of the liver is exceeded and hypoproteinemia, accompanied by anasarca, develops to cause the nephrotic syndrome. The massive urine protein loss is due to increased permeability of glomeruli to protein. The permeability increase may be minimal so that only albumin and other smaller molecular weight proteins are selectively filtered (selective nephrosis, as in Minimal Change Disease) or may be greater so that larger proteins are also filtered (nonselective nephrosis, as in membranous golmerulonephritis) as is the case in the example shown. Alpha-2-macroglobulin is sufficiently large so that it is not filtered and increased synthesis (from the general hepatic protein synthesis) causes its accumulation. Lipoproteins are also sufficiently large to accumulate and hyperlipidemia is a characteristic of the nephrotic syndrome, although lipoproteins are not stained with the protein stain used in visualizing proteins.

Alpha-1-Antitrypsin Deficiency --

A genetic defect causes a deficiency of alpha-1-antitrypsin. The antiprotease deficiency results in a propensity to develop emphysema. Since alpha-1-antitrypsin is the major component of the alpha-1 band, deficiency is suggested by a reduced alpha-1 band. Deficiency is confirmed by specific immunochemical quantification.

Acute Inflammation

The alpha-1 and alpha-2 bands are increased during the inflammatory response from increased hepatic synthesis of acute phase reactant proteins.

Chronic Inflammation --

Immunoglobulin synthesis by antigen activated B lymphocytes transformed to plasma cells is demonstrated by the increased polyclonal gamma band.

Immunoglobulin Deficiency --

Deficient immunoglobulin synthesis is revealed by a markedly diminished gamma band. Effected individuals are prone to recurrent infection.

Monoclonal Gammopathy --

An unusually sharp band in the gamma region strongly suggests the presence of a homogeneous immunoglobulin and, thus, the malignant proliferation of plasma cells from a single cell (multiple myeloma) in contrast to the broad, heterogeneous, or polyclonal, gamma band as exhibited above in chronic inflammation from immunoglobulin synthesis by many different clones of plasma cells. Homogeneous immunoglobulins are also found in Waldenstrom's macroglobulinemia (where the sharp gamma band is always IgM). Specimens which exhibit a narrow gamma band are further examined by immunofixation electrophoresis as described below

ImmunoFixation Electrophoresis
Immunofixation electrophoresis (IFE) is used to demonstrate that a narrow gamma band is due to a homogeneous immunoglobulin. IFE has superceded immunoelectrophoresis, results from which are considerably more difficult to interpret, for the purpose of evaluating monoclonal gammopathy.

Serum IFE

In the illustration to the left, 6 replicates of the specimen are loaded on to separate lanes of an IFE gel. Following electrophoresis, the protein in each lane is stained differently. The first lane (SP) is stained for total protein. The protein in each of the other lanes is "stained" with specific antisera for immunoglobulin heavy and light chains, respectively, as illustrated in the figure. The finding of the preponderance of only one light chain associated with a predominantly staining heavy chain confirms the molecular homogeneity of the immunoglobulin and also provides identification. IFE identifies the narrow band as monoclonal IgG, lambda.
Sometimes malignant plasma cells synthesize excess light chains and less frequently only light chains are synthesized. Almost never is excess heavy chain or only heavy chain synthesized. Excess free lambda light chain is exhibited in the illustration above. A greater amount of free light chain is found in an IFE of an accompanying urine specimen which is displayed below.
Polyclonal Gammaopathy.
An IFE of a serum specimen with a polyclonal gamma increase is shown for the sake of comparison.
Broad bands are present for both IgG and IgA and corresponding kappa and lambda light chain bands. The light chains appear to be present at the normal kappa/lambda ratio of about 2.
Urine IFE
Free light chains are readily filtered by the glomeruli and often are not detectable in serum specimens. Excess light chain synthesis results in proteinuria, which exhibits a narrow band upon electrophoresis. The identity of the narrow band is determined by IFE and is here seen to be free lambda light chain. (A trace of monoclonal IgG,lambda is also present in the urine specimen). The bottom-most band is albumin.

last updated on January 22, 2001.