Antigens and Antibodies

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Antigens and Antibodies

Learning Objectives

At the conclusion of this chapter, the reader should be able to:

• Define the terms antigen and antibody.

• Compare the characteristics of major histocompatibility complex (MHC) classes I and II.

• Name and compare the characteristics of each of the five immunoglobulin classes.

• Draw and describe a typical immunoglobulin G (IgG) molecular structure.

• Name the four phases of an antibody response.

• Describe the characteristics of a primary and secondary (anamnestic) response.

• Compare the terms antibody avidity and antibody affinity.

• Describe the method of production of a monoclonal antibody.

• Analyze a case study related to antigens or antibodies.

• Correctly answer case study related multiple choice questions.

• Be prepared to participate in a discussion of critical thinking questions.

• Describe the principle and agglutination reactions in ABO blood grouping.

• Describe the principle, expected results, reference values, and clinical interpretation of the serum protein electrophoresis procedure.

• Correctly answer end of chapter review questions.

Key Terms

Antigen Characteristics

General Characteristics of Immunogens and Antigens

An immune response is triggered by immunogens, macromolecules capable of triggering an adaptive immune response by inducing the formation of antibodies or sensitized T cells in an immunocompetent host (a host capable of recognizing and responding to a foreign antigen). Immunogens can specifically react with corresponding antibodies or sensitized T lymphocytes. In contrast, an antigen is a substance that stimulates antibody formation and has the ability to bind to an antibody or a T lymphocyte antigen receptor but may not be able to evoke an immune response initially. For example, lower molecular weight particles, haptens, can bind to an antibody but must be attached to a macromolecule as a carrier to stimulate a specific immune response. In reality, all immunogens are antigens but not all antigens are immunogens. The two terms, immunogens and antigens, are frequently used interchangeably without making a distinction between the two terms.

Foreign substances can be immunogenic or antigenic (capable of provoking a humoral and/or cell-mediated immune response) if their membrane or molecular components contain(s) structures recognized as foreign by the immune system. These structures are called antigenic determinants, or epitopes. An epitope, as part of an antigen, reacts specifically with an antibody or T lymphocyte receptor.

Not all surfaces act as antigenic determinants. Only prominent determinants on the surface of a protein are normally recognized by the immune system and some of these are much more immunogenic than others. An immune response is directed against specific determinants and resultant antibodies will bind to them, with much of the remaining molecule being immunogenic.

The cellular membrane of mammalian cells consists chemically of proteins, phospholipids, cholesterol, and traces of polysaccharide. Polysaccharides (carbohydrates) in the form of glycoproteins or glycolipids can be found attached to the lipid and protein molecules of the membrane. When antigen-bearing cells, such as red blood cells (RBCs), from one person, a donor, are transfused into another person, a recipient, they can be immunogenic. Outer surfaces of bacteria, such as the capsule or the cell wall, as well as the surface structures of other microorganisms, can also be immunogenic.

Cellular antigens of importance to immunologists include histocompatibility antigens, autoantigens, and blood group antigens (see later, “ABO Blood Grouping Procedure”). The normal immune system responds to foreignness by producing antibodies. For this reason, microbial antigens are also important to immunologists in the study of the immunologic manifestations of infectious disease.

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).

image
Figure 2-1 Genetic organization of MHC (HLA) antigen. LMP, Large multifunctional protease; TAP, transporter associated with antigen presentation. (From Nairn R, Helbert M: Immunology for medical students, ed 2, St Louis, 2007, Mosby.)

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
image
Figure 2-2 Structure of class I and class II MHC molecules.
The schematic diagrams (left) and models (right) of the crystal structures of class I and class II MHC molecules illustrate the domains of the molecules and the fundamental similarities between them. Both types of MHC molecules contain peptide-binding clefts and invariant portions that bind CD8 (the α3 domain of class I) or CD4 (the β2 domain of class II). β2m, β2-Microglobulin. (From Abbas AK, Lichtman AH: Basic immunology: functions and disorders of the immune system, updated edition, ed 3, Philadelphia, 2011, Saunders; crystal structures courtesy Dr. P. Bjorkman, California Institute of Technology, Pasadena, Calif.)

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.

Chemical Nature of Antigens

Antigens, or immunogens, are usually large organic molecules that are proteins or large polysaccharides and, rarely, if ever, lipids. Antigens, especially cell surface or membrane-bound antigens, can be composed of combinations of biochemical classes (e.g., glycoproteins, glycolipids). For example, histocompatibility HLAs are glycoprotein in nature and are found on the surface membranes of nucleated body cells composed of solid tissue and most circulating blood cells (e.g., granulocytes, monocytes, lymphocytes, thrombocytes).

Proteins are excellent antigens because of their high molecular weight and structural complexity. Lipids are considered inferior antigens because of their relative simplicity and lack of structural stability. However, when lipids are linked to proteins or polysaccharides, they may function as antigens. Nucleic acids are poor antigens because of relative simplicity, molecular flexibility, and rapid degradation. Anti–nucleic acid antibodies can be produced by artificially stabilizing them and linking them to an immunogenic carrier. Carbohydrates (polysaccharides) by themselves are considered too small to function as antigens. In the case of erythrocyte blood group antigens, protein or lipid carriers may contribute to the necessary size and the polysaccharides present in the form of side chains confer immunologic specificity.

Physical Nature of Antigens

Important factors in the effective functioning of antigens include foreignness, degradability, molecular weight (MW), structural stability, and complexity.

General Characteristics of Antibodies

Antibodies are specific proteins referred to as immunoglobulins. Many antibodies can be isolated in the gamma globulin fraction of protein by electrophoresis separation (Fig. 2-3). The term immunoglobulin (Ig) has replaced gamma globulin because not all antibodies have gamma electrophoretic mobility. Antibodies can be found in blood plasma and in many body fluids (e.g., tears, saliva, colostrum).

image
Figure 2-3 Tracing of the electrophoretic pattern of normal serum. (Adapted from Kaplan LA, Pesce AJ, Kazmierczak SC, editors: Clinical chemistry: theory, analysis, correlation, ed 4, St Louis, 2003, Mosby.)

The primary function of an antibody in body defenses is to combine with antigen, which may be enough to neutralize bacterial toxins or some viruses. A secondary interaction of an antibody molecule with another effector agent (e.g., complement) is usually required to dispose of larger antigens (e.g., bacteria).

Determining Ig concentration can be of diagnostic significance in infectious and autoimmune diseases. Test methods to detect the presence and concentration of immunoglobulins are discussed in Part II and in chapters relating to specific diseases.

Immunoglobulin (Ig) Classes

Five distinct classes of immunoglobulin molecules are recognized in most higher mammals—IgM, IgG, IgA, IgD, and IgE. These Ig classes differ from each other in characteristics such as MW and sedimentation coefficients (Table 2-2).

Table 2-2

Characteristics of Immunoglobulin Classes

  IgM IgG IgA IgE IgD
Molecular weight (daltons, Da) 900,000 160,000 360,000 200,000 160,000
Sedimentation coefficient (Σ) 19 7 11 8 7
Carbohydrate (%) 12 8 7 12 12
Subclasses — IgG1-4 α1, α2 — —
Serum concentration, adults (mg/mL) 1.5 13.5 3.5 0.05 Trace
Serum half-life (days)∗ 5 23 6 2.5 3

image

∗Half life (days) = the amount of time to reach ½ activity concentration. Serum values are average concentrations in normal, healthy individuals.

Adapted from Peakman M, Vergani D: Basic and clinical immunology, St Louis, 2009, Elsevier, p 41.

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).

image
Figure 2-4 Immunoglobulin concentration in newborns, infants, and children. (Adapted from Bauer JD: Clinical laboratory methods, ed 9, St Louis, 1982, Mosby.)

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:

Antibody Structure

Antibodies exhibit diversity among the different classes, which suggests that they perform different functions in addition to their primary function of antigen binding. Essentially, each Ig molecule is bifunctional; one region of the molecule involves binding to antigen, and a different region mediates binding of the immunoglobulin to host tissues, including cells of the immune system and the first component (C1q) of the classic complement system.

The primary core of an antibody consists of the sequence of amino acid residues linked by the peptide bond. All antibodies have a common, basic polypeptide structure, with a three-dimensional configuration. The polypeptide chains are linked by covalent and noncovalent bonds, which produce a unit composed of a four-chain structure based on pairs of identical heavy and light chains. IgG, IgD, and IgE occur only as monomers of the four-chain unit, IgA occurs in both monomeric and polymeric forms, and IgM occurs as a pentamer with five four-chain subunits linked together.

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.

image
Figure 2-5

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