Histocompatibility Antigens

Nucleated cells such as leukocytes and tissues possess many cell surface–protein antigens that readily provoke an immune response if transferred into a genetically different (allogenic) individual of the same species. Some of these antigens, which constitute the major histocompatibility complex (MHC) (see Color Plate 2), are more potent than others in provoking an immune response. The MHC is referred to as the human leukocyte antigen (HLA) system in humans because its gene products were originally identified on white blood cells (WBCs, leukocytes). These antigens are second only to the ABO antigens in influencing the survival or graft rejection of transplanted organs. HLAs are the subject of numerous scientific investigations because of the strong association between individual HLAs and immunologic disorders (see Chapter 31 for more discussion of the MHC).

Major Histocompatibility Complex Regions

The MHC is divided into four major regions (Fig. 2-1)—D, B, C, and A. The A, B, and C regions are the classic or class Ia genes that code for class I molecules. The D region codes for class II molecules. Class I includes HLA-A, HLA-B, and HLA-C. The three principal loci (A, B, and C) and their respective antigens are numbered, for example, as 1, 2, 3. The class II gene region antigens are encoded in the HLA-D region and can be subdivided into three families, HLA-DR, HLA-DC (DQ), and HLA-SB (DP).

Classes of HLA Molecules

Structurally, there are two classes of HLA molecules, class I and class II (Table 2-1). Both class I and class II antigens function as targets of T lymphocytes (see Chapter 4 for a further discussion of lymphocytes) that regulate the immune response (Fig. 2-2). Class I molecules regulate interaction between cytolytic T cells and target cells and class II molecules restrict the activity of regulatory T cells. Thus, class II molecules regulate the interaction between helper T cells and antigen-presenting cells (APCs). Cytotoxic T cells directed against class I antigens are inhibited by CD8 cells; cytotoxic T cells directed against class II antigens are inhibited by CD4 cells. Many genes in the class I and class II gene families have no known function.

Table 2-1

Comparison of MHC Class I and Class II

	Class I	Class II
Loci	HLA-A, -B, and -C	HLA-DN, -DO, -DP, -DQ, and -DR
Distribution	Most nucleated cells	B lymphocytes, macrophages, other antigen-presenting cells, activated T lymphocytes
Function	To present endogenous antigen to cytotoxic T lymphocytes	To present endogenous antigen to helper T lymphocytes

Autoantigens

The evolution of a recognition system that can recognize and destroy nonself material must also have safeguards to prevent damage to self antigens. The body’s immune system usually exercises tolerance to self antigens but, in some situations, antibodies may be produced in response to normal self antigens. This failure to recognize self antigens can result in autoantibodies directed at hormones, such as thyroglobulin (see Chapter 28).

Blood Group Antigens

Blood group substances are widely distributed throughout the tissues, blood cells, and body fluids. When foreign RBC antigens are introduced to a host, a transfusion reaction or hemolytic disease of the fetus and newborn can result (see Chapter 26). In addition, certain antigens, especially those of the Rh system, are integral structural components of the erythrocyte (RBC) membrane. If these antigens are missing, the erythrocyte membrane is defective and results in hemolytic anemia. When antigens do not form part of the essential membrane structure (e.g., A, B, and H antigens), the absence of antigen has no effect on membrane integrity.

Adjuvant

The response to immunization can be enhanced by a number of agents, collectively called adjuvants. One of the best-known emulsifying agents in vaccine studies is Freund’s complete adjuvant. An adjuvant is a substance, distinct from antigen, that enhances T cell activation by promoting the accumulation of APCs at a site of antigen exposure and by enhancing the expression of costimulators and cytokines by the APCs.

Foreignness

Foreignness is the degree to which antigenic determinants are recognized as nonself by an individual’s immune system. The immunogenicity of a molecule depends to a great extent on its degree of foreignness. For example, if a transplant recipient receives a donor organ with several major HLA differences, the organ is perceived as foreign and is subsequently rejected by the recipient. Normally, an individual’s immune system does not respond to self antigens.

Degradability

For an antigen to be recognized as foreign by an individual’s immune system, sufficient antigens to stimulate an immune response must be present. Foreign molecules are rapidly destroyed and thus cannot provide adequate antigenic exposure. In the case of vaccination, an adequate dose of vaccine at appropriate intervals must be administered for an immune response to be stimulated.

Molecular Weight

The higher the MW, the better the molecule will function as an antigen. The number of antigenic determinants on a molecule is directly related to its size. For example, proteins are effective antigens because of a large MW.

Although large foreign molecules (MW 10,000 daltons [Da]) are better antigens, haptens, which are tiny molecules, can bind to a larger carrier molecule and behave as antigens. If a hapten is chemically linked to a large molecule, a new surface structure is formed on the large molecule, which may function as an antigenic determinant.

Structural Stability

If a molecule is an effective antigen, structural stability is mandatory. If a structure is unstable (e.g., gelatin), the molecule will be a poor antigen. Similarly, totally inert molecules are poor antigens. Ther structural stability of an antigen is important in cases where the goal is to elicit a patient antibody response when adminstering a vaccine.

Complexity

The more complex an antigen, the greater is its effectiveness. Complex proteins are better antigens than large repeating polymers such as lipids, carbohydrates, and nucleic acids, which are relatively poor antigens.

Immunoglobulin M

Immunoglobulin M accounts for about 10% of the Ig pool and is largely confined to the intravascular pool because of its large size. This antibody is produced early in an immune response and is largely confined to the blood. IgM is effective in agglutination and cytolytic reactions. In humans, IgM is found in smaller concentrations than IgG or IgA. The molecule has five individual heavy chains, with an MW of 65,000 Da; the whole molecule has an MW of 900,000 Da and sedimentation coefficient, Σ, of 19.

Normal values of IgM are 60 to 250 mg/dL (70 to 290 IU/mL) for males and 70 to 280 mg/dL (80 to 320 IU/mL) for females. At 4 months of age, 50% of the adult level is present; adult levels are reached by 8 to 15 years. Cord blood contains greater than 20 mg/dL. IgM is usually undetectable in cerebrospinal fluid (CSF).

IgM is decreased in primary (genetically determined) Ig disorders as well as secondary Ig deficiencies (acquired disorders associated with certain diseases). IgM can be increased in the following conditions:

Immunoglobulin G

The major immunoglobulin in normal serum is IgG. It diffuses more readily than other immunoglobulins into the extravascular spaces and neutralizes toxins or binds to microorganisms in extravascular spaces. IgG can cross the placenta. In addition, when IgG complexes are formed, complement can be activated. IgG accounts for 70% to 75% of the total Ig pool. It is a 7S molecule, with an MW of approximately 150,000 Da. One of the subclasses, IgG3, is slightly larger (170,000 Da) than the other subclasses.

Normal human adult serum values of IgG are 800 to 1800 mg/dL (90 to 210 IU/mL). In infants 3 to 4 months old, the IgG level is approximately 350 to 400 mg/dL (40 to 45 IU/mL), gradually increasing to 700 to 800 mg/dL (80 to 90 IU/mL) by the end of the first year of life (Fig. 2-4). The average adult level is achieved before age 16 years. Other body fluids containing IgG include cord blood (800 to 1800 mg/dL) and CSF (2 to 4 mg/dL).

Decreased levels of IgG can be manifested in primary (genetic) or secondary (acquired) Ig deficiencies. Significant increases of IgG are seen in the following conditions:

Immunoglobulin A

Immunoglobulin A represents 15% to 20% of the total circulatory Ig pool. It is the predominant immunoglobulin in secretions such as tears, saliva, colostrum, milk, and intestinal fluids. IgA is synthesized largely by plasma cells located on body surfaces. If produced by cells in the intestinal wall, IgA may pass directly into the intestinal lumen or diffuse into the blood circulation. As IgA is transported through intestinal epithelial cells or hepatocytes, it binds to a glycoprotein called the secretory component. The secretory piece protects IgA from digestion by gastrointestinal proteolytic enzymes. It forms a complex molecule termed secretory IgA, which is critical in protecting body surfaces against invading microorganisms because of its presence in seromucous secretions (e.g., tears, saliva, nasal fluids, colostrum).

IgA monomer is present in relatively high concentrations in human serum; it has a concentration of 90 to 450 mg/dL (55 to 270 IU/mL) in normal adult humans. At the end of the first year of life, 25% of the adult IgA level is reached, and 50% at 3.5 years of age. The average adult level is attained by age 16 years. IgA concentration in cord blood is greater than 1 mg/dL; CSF contains 0.1 to 0.6 mg/dL of IgA.

IgA is decreased in primary or secondary Ig deficiencies. Significant increases in serum IgA concentration are associated with the following:

Immunoglobulin D

Immunoglobulin D is found in very low concentrations in plasma, accounting for less than 1% of the total Ig pool. IgD is extremely susceptible to proteolysis and is primarily a cell membrane Ig found on the surface of B lymphocytes in association with IgM.

Immunoglobulin E

Immunoglobulin E is a trace plasma protein found in the blood plasma of unparasitized individuals (MW, 188,000 Da). IgE is crucial because it mediates some types of hypersensitivity (allergic) reactions, allergies, and anaphylaxis and is generally responsible for an individual’s immunity to invading parasites. The IgE molecule is unique in that it binds strongly to a receptor on mast cells and basophils and, together with antigen, mediates the release of histamines and heparin from these cells.

Typical Immunoglobulin Molecule

The basic unit of an antibody structure is the homology unit, or domain. A typical molecule has 12 domains, arranged in two heavy (H) and two light (L) chains, linked through cysteine residues by disulfide bonds so that the domains lie in pairs (Fig. 2-5). The antigen-binding portion of the molecule (N-terminal end) shows such heterogeneity that it is known as the variable (V) region; the remainder is composed of relatively constant amino acid sequences, the constant (C) region. Short segments of about 10 amino acid residues within the variable regions of antibodies (or T cell receptor [TCR] proteins) form loop structures called complementary-determining regions (CDRs). Three hypervariable loops, also called CDRs, are present in each antibody H chain and L chain. Most of the variability among different antibodies or TCRs is located within these loops.

The IgG molecule provides a classic model of antibody structure, appearing Y-shaped under electron microscopy (Fig. 2-6). If the molecule is studies by chemical treatment and the interchain disulfide bonds are broken, the molecule separates into four polypeptide chains. Light chains are small chains (25,000 Da) common to all Ig classes. The L chains are of two subtypes, kappa (κ) and lambda (λ), which have different amino acid sequences and are antigenically different. In humans, about 65% of Ig molecules have κ chains, whereas 35% have λ chains. The larger H chains (50,000 to 77,000 Da) extend the full length of the molecule.

A general feature of the Ig chains is their amino acid sequence. The first 110 to 120 amino acids of both L and H chains have a variable sequence and form the V region; the remainder of the L chains represents the C region, with a similar amino acid sequence for each type and subtype. The remaining portion of the H chain is also constant for each type and has a hinge region. The class and subclass of an Ig molecule are determined by its H-chain type.

Fab, Fc, and Hinge Molecular Components

A typical monomeric IgG molecule consists of three globular regions (two Fab regions and an Fc portion) linked by a flexible hinge region. If the molecule is digested with a proteolytic enzyme such as papain, it splits into three approximately equal-sized fragments (Fig. 2-7). Two of these fragments retain the ability to bind antigen and are called the antigen-binding fragments (Fab fragments). The third fragment, which is relatively homogeneous and is sometimes crystallizable, is called the Fc portion. If IgG is treated with another proteolytic enzyme, pepsin, the molecule separates somewhat differently. The Fc fragment is split into tiny peptides and thus is completely destroyed. The two Fab fragments remain joined to produce a fragment called F(ab)′2. This fragment possesses two antigen-binding sites. If F(ab)′2 is treated to reduce its disulfide bonds, it breaks into two Fab fragments, each of which has only one antigen-binding site. Further disruption of the interchain disulfide bonds in the Fab fragments shows that each contains a light chain and half of a heavy chain, which is called the Fd fragment.

Electron microscopy studies of IgG have revealed that the Fab regions of the molecule are mobile and can swing freely around the center of the molecule as if it were hinged. This hinge consists of a group of about 15 amino acids located between the C_H1 and C_H2 regions. The exact sequence of amino acids in the hinge is variable and unique for each Ig class and subclass. Because amino acids can rotate freely around peptide bonds, the effect of closely spaced proline amino acid residues is production of a so-called universal joint, around which the Ig chains can swing freely. A remarkable feature of the hinge region is the presence of a large number of hydrophilic and proline residues. The hydrophilic residues tend to open up this region and thus make it accessible to proteolytic cleavage with enzymes such as pepsin and papain. This region also contains all the interchain disulfide bonds except for IgD, which has no interchain links.

The IgM molecule is structurally composed of five basic subunits. Each subunit consists of two κ or two λ light chains and two mu (µ) heavy chains. The individual monomers of IgM are linked together by disulfide bonds in a circular fashion (Fig. 2-8). A small, cysteine-rich polypeptide, the J chain, must be considered an integral part of the molecule. IgM has carbohydrate residues attached to the C_H3 and C_H4 domains. The site for complement activation by IgM is located on this C_H4 region. IgM is more efficient than IgG in activities such as complement cascade activation and agglutination.

Immunoglobulin A

In humans, more than 80% of IgA occurs as a typical four-chain structure consisting of paired κ or λ chains and two heavy chains (Fig. 2-9). The basic four-chain monomer has an MW of 160,000 Da; however, in most mammals, plasma IgA occurs mainly as a dimer. In dimeric IgA, the molecules are joined by a J chain linked to the Fc regions. Secretory IgA exists mainly in the 11S dimeric form and has an MW of 385,000 Da (Fig. 2-10). This form of IgA is present in fluids and is stabilized against proteolysis when combined with another protein, the secretory component. In humans, variations in the heavy chains account for the subclasses IgA1 and IgA2.

Immunoglobulin D

The IgD molecule has an MW of 184,000 Da and consists of two κ or α light chains and two delta (δ) heavy chains (Fig. 2-11). It has no interchain disulfide bonds between its heavy chains and an exposed hinge region.

Immunoglobulin E

The IgE molecule is composed of paired κ or α light chains and two epsilon (ε) heavy chains (Fig. 2-12). It is unique in that its Fc region binds strongly to a receptor on mast cells and basophils and, together with antigen, mediates the release of histamines and heparin from these cells.

Isotype Determinants

The isotypic class of antigenic determinants is the dominant type found on the immunoglobulins of all animals of a species. The heavy-chain, constant region structures associated with the different classes and subclasses are termed isotypic variants. Genes for isotypic variants are present in all healthy members of a species. Determinants in this category include those specific for each Ig class, such as gamma (γ) for IgG, mu (µ) for IgM, and alpha (α) for IgA, as well as the subclass-specific determinants κ and λ.

Allotype Determinants

The second principal group of determinants is found on the immunoglobulins of some, but not all, animals of a species. Antibodies to these allotypes (alloantibodies) may be produced by injecting the immunoglobulins of one animal into another member of the same species. The allotypic determinants are genetically determined variations representing the presence of allelic genes at a single locus within a species. Typical allotypes in humans are the Gm specificities on IgG (Gm is a marker on IgG). In humans, five sets of allotypic markers have been found—Gm, Km, Mm, Am, and Hv.

Idiotype Determinants

A result of the unique structures on light and heavy chains, individual determinants characteristic of each antibody are called idiotypes. The idiotypic determinants are located in the variable part of the antibody associated with the hypervariable regions that form the antigen-combining site.

Primary Antibody Response

Although the duration and levels of antibody (titer) depend on the characteristics of the antigen and the individual, an IgM antibody response proceeds in the following four phases after a foreign antigen challenge (see Fig. 2-14):

Secondary (Anamnestic) Response

Subsequent exposure to the same antigenic stimulus produces an antibody response that exhibits the same four phases as the primary response (see Fig. 2-14). Repeated exposure to an antigen can occur many years after the initial exposure, but clones of memory cells will be stimulated to proliferate, with subsequent production of antibody by the individual. An anamnestic response differs from a primary response as follows:

An example of an anamnestic response can be observed in hemolytic disease, when an Rh-negative mother is pregnant with an Rh-positive baby (see Chapter 26). During the mother’s first exposure, the Rh-positive RBCs of the fetus leak into the maternal circulation and elicit a primary response. Subsequent pregnancies with an Rh-positive fetus will elicit a secondary (anamnestic) response.

Vaccination is the application of primary and second responses. Humans can become immune to microbial antigens through artificial and natural exposure. A vaccine is designed to provide artificially acquired active immunity to a specific disease (e.g., hepatitis B). Booster vaccine (repeated antigen exposure) allows for an anamnestic response, with an increase in antibody titer and clones of memory cells (see Chapter 16).

Antibody Affinity

Affinity is the initial force of attraction that exists between a single Fab site on an antibody molecule and a single epitope or determinant site on the corresponding antigen. The antigen is univalent and is usually a hapten. Several types of noncovalent bonds hold an epitope and binding site close together (see later, “Type of Bonding”).

Antibody Avidity

Each four-polypeptide–chain antibody unit has two antigen-binding sites, which allows them to be potentially multivalent in their reaction with an antigen. The functional combining strength of an antibody with its antigen is called avidity, in contrast to affinity, the binding strength between an antigenic determinant (epitope) and an antibody-combining site (Fig. 2-15). When a multivalent antigen combines with more than one of an antibody’s combining sites, the strength of the bonding is significantly increased. For the antigen and antibody to dissociate, all the antigen-antibody bonds must be broken simultaneously.

Decreased avidity can result when an antigen (e.g., hapten) has only one antigenic determinant (monovalent).

Immune Complexes

The noncovalent combination of antigen with its respective specific antibody is called an immune complex. An immune complex may be of the small (soluble) or large (precipitating) type, depending on the nature and proportion of antigen and antibody. Under conditions of antigen or antibody excess, soluble complexes tend to predominate. If equivalent amounts of antigen and antibody are present, a precipitate may form. However, all antigen-antibody complexes will not precipitate, even at equivalence.

Antibody can react with antigen that is fixed or localized in tissues or that is released or present in the circulation. Once formed in the circulation, the immune complex is usually removed by phagocytic cells through the interaction of the Fc portion of the antibody with complement and cell surface receptors.

Under normal circumstances, this process does not lead to pathologic consequences and it may be viewed as a major host defense against the invasion of foreign antigens. It is only in unusual circumstances that the immune complex persists as a soluble complex in the circulation, escapes phagocytosis, and is deposited in endothelial or vascular structures—where it causes inflammatory damage, the principal characteristic of immune complex disease—or in organs (e.g., kidney), or inhibits useful immunity (e.g., tumors, parasites). The level of circulating immune complex is determined by the rate of formation, rate of clearance and, most importantly, nature of the complex formed. Detection of immune complexes and identification of the associated antigens are important to the clinical diagnosis of immune complex disorders.

Types of Bonding

Bonding of an antigen to an antibody results from the formation of multiple, reversible, intermolecular attractions between an antigen and amino acids of the binding site. These forces require proximity of the interacting groups. The optimum distance separating the interacting groups varies for different types of bond; however, all these bonds act only across a very short distance and weaken rapidly as that distance increases.

The bonding of antigen to antibody is exclusively noncovalent. The attractive force of noncovalent bonds is weak compared with that of covalent bonds, but the formation of multiple noncovalent bonds produces considerable total binding energy. The strength of a single antigen-antibody bond (antibody affinity) is produced by the summation of the attractive and repulsive forces. The four types of noncovalent bonds involved in antigen-antibody reactions are hydrophobic bonds, hydrogen bonds, van der Waals forces, and electrostatic forces.

Goodness of Fit

The strongest bonding develops when antigens and antibodies are close to each other and when the shapes of the antigenic determinants and the antigen-binding site conform to each other. This complementary matching of determinants and binding sites is referred to as goodness of fit (Fig. 2-16).

A good fit will create ample opportunities for the simultaneous formation of several noncovalent bonds and few opportunities for disruption of the bond. If a poor fit exists, repulsive forces can overpower any small forces of attraction. Variations from the ideal complementary shape will produce a decrease in the total binding energy because of increased repulsive forces and decreased attractive forces. Goodness of fit is important in determining the binding of an antibody molecule for a particular antigen.

Detection of Antigen-Antibody Reactions

In vitro tests detect the combination of antigens and antibodies. Agglutination is the process whereby particulate antigens (e.g., cells) aggregate to form larger complexes in the presence of a specific antibody. Agglutination tests are widely used in immunology to detect and measure the consequences of antigen-antibody interaction. Other tests include the following:

The principles of immunologic methods are discussed in Part II of this text. Detection and quantitation of immunoglobulins is important in the laboratory investigation of infectious diseases and immunologic disorders (Table 2-5).

Influence of Antibody Types on Agglutination

Immunoglobulins are relatively positively charged and, after sensitization or coating of particles, they reduce the zeta potential, which is the difference in electrostatic potential between the net charge at the cell membrane and the charge at the surface of shear (see Fig. 10-4). Antibodies can bridge charged particles by extending beyond the effective range of the zeta potential, which results in the erythrocytes closely approaching each other, binding, and agglutinating.

Antibodies differ in their ability to agglutinate. IgM-type antibodies, sometimes referred to as complete antibodies, are more efficient than IgG or IgA antibodies in exhibiting in vitro agglutination when the antigen-bearing erythrocytes are suspended in physiologic saline (0.9% sodium chloride solution). Antibodies that do not exhibit visible agglutination of saline-suspended erythrocytes, even when bound to the cell’s surface membrane, are considered to be nonagglutinating antibodies and have been called incomplete antibodies. Incomplete antibodies may fail to exhibit agglutination because the antigenic determinants are located deep within the surface membrane or may show restricted movement in their hinge region, causing them to be functionally monovalent.

Discovery of the Technique

In 1975, Köhler, Milstein, and Jerne discovered how to fuse lymphocytes to produce a cell line that was both immortal and a producer of specific antibodies. These scientists were awarded the Nobel Prize in Physiology and Medicine in 1984 for developing this hybridoma (cell hybrid) from different lines of cultured myeloma cells (plasma cells derived from malignant tumor strains). To induce the cells to fuse, they used Sendai virus, an influenza virus that characteristically causes cell fusion. Initially, the scientists immunized donors with sheep erythrocytes to provide a marker for the normal cells. The hybrids were tested to determine whether they still produced antibodies against the sheep erythrocytes. Köhler discovered that some of the hybrids were manufacturing large quantities of specific anti–sheep erythrocyte antibodies.

Hybrid cells secrete the antibody that is characteristic of the parent cell (e.g., anti–sheep erythrocyte antibodies). The multiplying hybrid cell culture is a hybridoma. Hybridoma cells can be cloned. The immunoglobulins derived from a single clone of cells are termed monoclonal antibodies (MAbs).

Monoclonal Antibody Production

Modern methods for producing MAbs are refinements of the original technique. Basically, the hybridoma technique enables scientists to inoculate crude antigen mixtures into mice and then select clones producing specific antibodies against a single cell surface antigen (Fig. 2-17). The process of producing MAbs takes 3 to 6 months.

Mice are immunized with a specific antigen; several doses are given to ensure a vigorous immune response. After 2 to 4 days, spleen cells are mixed with cultured mouse myeloma cells. Myeloma parent cells that lack the enzyme, hypoxanthine phosphoribosyl transferase, are selected. Mouse myeloma cell lines usually do not secrete immunoglobulins, thus simplifying the purification process.

Polyethylene glycol (PEG) rather than Sendai virus is added to the cell mixture to promote cell membrane fusion. Only 1 in 200,000 spleen cells actually forms a viable hybrid with a myeloma cell. Normal spleen cells do not survive in culture. The fused cell mixture is placed in a medium containing hypoxanthine, aminopterin, and thymidine (HAT medium). Aminopterin is a drug that prevents myeloma cells from making their own purines and pyrimidines; they cannot use hypoxanthine from the medium, so they die.

Hybrids resulting from the fusion of spleen cells and myeloma cells contain transferase provided by the normal spleen cells. Consequently, the hybridoma cells are able to use the hypoxanthine and thymidine in the culture medium and survive. They divide rapidly in HAT medium, doubling in number every 24 to 48 hours. About 300 to 500 hybrids can be generated from the cells of a single mouse spleen, although not all will be making the desired antibodies. After the hybridomas have been growing for 2 to 4 weeks, the supernatant is tested for specific antibody using methods such as ELISA. Clones that produce the desired antibody are grown in mass culture and recloned to eliminate non–antibody-producing cells.

Antibody-producing clones lose their ability to synthesize or secrete antibody after being cultured for several months. Hybridoma cells usually are frozen and stored in small aliquots. The cells may then be grown in mass culture or injected intraperitoneally into mice. Because hybridomas are tumor cells, they grow rapidly and induce the effusion of large quantities of fluid into the peritoneal cavity. This ascites fluid is rich in MAbs and can be easily harvested.

Uses of Monoclonal Antibodies

The greatest impact of MAbs in immunology has been on the analysis of cell membrane antigens. Because they have a single specificity rather than the range of antibody molecules present in the serum, MAbs have multiple clinical applications, including the following:

Results

The fastest moving band, and normally the most prominent, is the albumin band found closest to the anodic edge of the plate. The faint band next to this is alpha-1 globulin, followed by alpha-2, beta, and gamma globulins. Prealbumin is seldom visible with this system.

Reference Values

Each laboratory should establish its own range. The following reference values are for illustrative purposes only.

Clinical Interpretation

Electrophoresis is used to identify the presence or absence of aberrant proteins and to determine when different groups of proteins are increased or decreased in serum or urine. It is frequently ordered to detect and identify monoclonal proteins—excessive production of one specific immunoglobulin. Protein and immunofixation electrophoresis are ordered to help detect, diagnose, and monitor the course and treatment of conditions associated with these abnormal proteins (e.g., multiple myeloma).