Review of Medical Microbiology and Immunology, 13th Edition

59. Antibodies



Monoclonal Antibodies

Immunoglobulin Structure

Immunoglobulin Classes






Isotypes, Allotypes, & Idiotypes

Immunoglobulin Genes

Immunoglobulin Class Switching (Isotype Switching)

Allelic Exclusion

Catalytic Antibody

Self-Assessment Questions

Practice Questions: USMLE & Course Examinations


Antibodies are globulin proteins (immunoglobulins [Ig]) that react specifically with the antigen that stimulated their production. They make up about 20% of the protein in blood plasma. Blood contains three types of globulins, alpha, beta, and gamma, based on their electrophoretic migration rate. Antibodies are gamma globulins. There are five classes of antibodies: IgG, IgM, IgA, IgD, and IgE. Antibodies are subdivided into these five classes based on differences in their heavy chains.

The most important functions of antibodies are to neutralize toxins and viruses, to opsonize microbes so they are more easily phagocytosed, to activate complement, and to prevent the attachment of microbes to mucosal surfaces. The specific antibody classes that mediate these functions are described in Table 59–1. In addition to these functions, antibodies have a catalytic (enzymatic) capability that is described in a separate section at the end of this chapter.

TABLE 59–1 Properties of Human Immunoglobulins



Antibodies that arise in an animal in response to typical antigens are heterogeneous, because they are formed by several different clones of plasma cells (i.e., they are polyclonal). Antibodies that arise from a single clone of cells (e.g., in a plasma cell tumor [myeloma])1 are homogeneous (i.e., they are monoclonal).

Monoclonal antibodies also can be made in the laboratory by fusing a myeloma cell with an antibody-producing cell (Figure 59–1; also see box “Hybridomas & Monoclonal Antibodies”). Such hybridomas produce virtually unlimited quantities of monoclonal antibodies that are useful in diagnostic tests and in research (see box “Hybridomas & Monoclonal Antibodies”).


FIGURE 59–1 Production of monoclonal antibodies.


Immunoglobulins are glycoproteins made up of light (L) and heavy (H) polypeptide chains. The terms light and heavy refer to molecular weight; light chains have a molecular weight of about 25,000, whereas heavy chains have a molecular weight of 50,000 to 70,000. The simplest antibody molecule has a Y shape (Figure 59–2) and consists of four polypeptide chains: two H chains and two L chains. The four chains are linked by disulfide bonds. An individual antibody molecule always consists of identical H chains and identical L chains. This is primarily the result of two phenomena: allelic exclusion (see page 514) and regulation within the B cell, which ensure the synthesis of either kappa (κ) or lambda (λ) L chains, but not both.


FIGURE 59–2 Structure of immunoglobulin G (IgG). The Y-shaped IgG molecule consists of two light chains and two heavy chains. Each light chain consists of a variable region and a constant region. Each heavy chain consists of a variable region and a constant region that is divided into three domains: CH1, CH2, and CH3. The CH2 domain contains the complement-binding site, and the CH3 domain is the site of attachment of IgG to receptors on neutrophils and macrophages. The antigen-binding site is formed by the variable regions of both the light and heavy chains. The specificity of the antigen-binding site is a function of the amino acid sequence of the hypervariable regions (see Figure 59–3). (Modified and reproduced with permission from Brooks GF et al. Medical Microbiology. 20th ed. Originally published by Appleton & Lange. Copyright 1995 McGraw-Hill.)

L and H chains are subdivided into variable and constant regions. The regions are composed of three-dimensionally folded, repeating segments called domains. An L chain consists of one variable (VL) and one constant (CL) domain. Most H chains consist of one variable (VH) and three constant (CH) domains. (IgG and IgA have three CH domains, whereas IgM and IgE have four.) Each domain is approximately 110 amino acids long. The variable regions of both the light and heavy chain are responsible for antigen-binding, whereas the constant region of the heavy chain is responsible for various biologic functions (e.g., complement activation and binding to cell surface receptors). The complement binding site is in the CH2 domain. The constant region of the light chain has no known biologic function.


Hybridoma cells have the remarkable ability to produce large quantities of a single molecular species of immunoglobulin. These immunoglobulins, which are known as monoclonal antibodies, are called “monoclonal” because they are made by a clone of cells that arose from a single cell. Note, however, that this single cell is, in fact, formed by the fusion of two different cells (i.e., it is a hybrid), hence the term hybridoma. Hybridoma cells are made in the following manner: (1) An animal (e.g., a mouse) is immunized with the antigen of interest. (2) Spleen cells from this animal are grown in a culture dish in the presence of mouse myeloma cells. The myeloma cells have two important attributes: They grow indefinitely in culture, and they do not produce immunoglobulins. (3) Fusion of the cells is encouraged by adding certain chemicals (e.g., polyethylene glycol). (4) The cells are grown in a special culture medium (HAT medium) that supports the growth of the fused, hybrid cells but not of the “parental” cells. (5) The resulting clones of cells are screened for the production of antibody to the antigen of interest.

Chimeric monoclonal antibodies consisting of mouse variable regions and human constant regions are being made for use in treating human diseases such as leukemia. The advantages of the human constant chain are that human complement is activated (whereas it is not if the constant region is mouse-derived) and that antibodies against the monoclonal antibody are not formed (whereas antibodies are formed if the constant region is mouse-derived). The advantage of the mouse variable region is that it is much easier to obtain monoclonal antibodies against, for example, a human tumor antigen by inoculating a mouse with the tumor cells. Chimeric antibodies can kill tumor cells either by complement-mediated cytotoxicity or by delivering toxins (e.g., diphtheria toxin) specifically to the tumor cell.

Monoclonal antibodies are now used in a variety of clinical situations, such as immunosuppression related to organ transplants, treatment of autoimmune disease, treatment of cancer, and the prevention of infectious disease. Table 62–2 describes these monoclonal antibodies, their cellular targets, and their clinical use.

The variable regions of both L and H chains have three extremely variable (hypervariable) amino acid sequences at the amino-terminal end that form the antigen-binding site (Figure 59–3). Only 5 to 10 amino acids in each hypervariable region form the antigen-binding site. Antigen–antibody binding involves electrostatic and van der Waals’ forces and hydrogen and hydrophobic bonds rather than covalent bonds. The remarkable specificity of antibodies is due to these hypervariable regions (see the discussion of idiotypes on page 512).


FIGURE 59–3 The antigen-binding site is formed by the hypervariable regions. A: Hypervariable regions on immunoglobulin G (IgG). B: Magnified view of antigen-binding site. (Modified and reproduced with permission from Stites DP, Terr A, Parslow T, eds. Basic & Clinical Immunology. 8th ed. Originally published by Appleton & Lange. Copyright 1994 McGraw-Hill.)

L chains belong to one of two types, κ (kappa) or λ (lambda), on the basis of amino acid differences in their constant regions. Both types occur in all classes of immunoglobulins (IgG, IgM, etc.), but any one immunoglobulin molecule contains only one type of L chain.2

The amino-terminal portion of each L chain participates in the antigen-binding site. H chains are distinct for each of the five immunoglobulin classes and are designated γ, α, μ, ε, and δ (Table 59–2). The amino-terminal portion of each H chain participates in the antigen-binding site; the carboxy terminal forms the Fc fragment, which has the biologic activities described earlier and in Table 59–2.

TABLE 59–2 Important Functions of Immunoglobulins


If an antibody molecule is treated with a proteolytic enzyme such as papain, peptide bonds in the “hinge” region are broken, producing two identical Fab fragments, which carry the antigen-binding sites, and one Fc fragment, which is involved in placental transfer, complement fixation, attachment site for various cells, and other biologic activities (Figure 59–2).



Each IgG molecule consists of two L chains and two H chains linked by disulfide bonds (molecular formula H2L2). Because it has two identical antigen-binding sites, it is said to be divalent. There are four subclasses, IgG1–IgG4, based on antigenic differences in the H chains and on the number and location of disulfide bonds. IgG1 makes up most (65%) of the total IgG. IgG2 antibody is directed against polysaccharide antigens and is an important host defense against encapsulated bacteria.

IgG is the predominant antibody in the secondary response and constitutes an important defense against bacteria and viruses (Table 59–1). IgG is the only antibody to cross the placenta; only its Fc portion binds to receptors on the surface of placental cells. It is therefore the most abundant immunoglobulin in newborns. IgG is one of the two immunoglobulins that can activate complement; IgM is the other (see Chapter 63).

IgG is the immunoglobulin that opsonizes. It can opsonize (i.e., enhance phagocytosis) because there are receptors for the γH chain on the surface of phagocytes. IgM does not opsonize directly, because there are no receptors on the phagocyte surface for the μH chain. However, IgM activates complement, and the resulting C3b can opsonize because there are binding sites for C3b on the surface of phagocytes. Note that there are four subclasses of IgG. Subclasses IgG1 and IgG3 are more effective opsonizers than are IgG2 and IgG4.

IgG has various sugars attached to the heavy chains, especially in the CH2 domain. The medical importance of these sugars is that they determine whether IgG will have a proinflammatory or anti-inflammatory effect. For example, if the IgG molecule has a terminal N-acetyl glucosamine, it is proinflammatory because it will bind to mannose-binding ligand and activate complement (see Chapter 63 and Figure 63–1). In contrast, if the IgG has a sialic acid side chain, then it will not bind and becomes anti-inflammatory. Thus IgG proteins specific for a single antigen that are made by a single plasma cell can, at various times, possess different properties depending on these sugar modifications.


IgA is the main immunoglobulin in secretions such as colostrum, saliva, tears, and respiratory, intestinal, and genital tract secretions. It prevents attachment of microorganisms (e.g., bacteria and viruses) to mucous membranes. Each secretory IgA molecule consists of two H2L2 units plus one molecule each of J (joining) chain3 and secretory component (Figure 59–4). The two heavy chains in IgA are α heavy chains.


FIGURE 59–4 Structure of serum immunoglobulin (Ig) A (A), secretory IgA (B), and IgM (C). Note that both IgA and IgM have a J chain but that only secretory IgA has a secretory component. (Reproduced with permission from Stites D, Terr A, Parslow T, eds. Basic & Clinical Immunology. 8th ed. Originally published by Appleton & Lange. Copyright 1994 McGraw-Hill.)

The secretory component is a polypeptide synthesized by epithelial cells that provides for IgA passage to the mucosal surface. It also protects IgA from being degraded in the intestinal tract. In serum, some IgA exists as monomeric H2L2.


IgM is the main immunoglobulin produced early in the primary response. It is present as a monomer on the surface of virtually all B cells, where it functions as an antigen-binding receptor.4 In serum, it is a pentamer composed of five H2L2 units plus one molecule of J (joining) chain (Figure 59–4). IgM has a μ heavy chain. Because the pentamer has 10 antigen-binding sites, it is the most efficient immunoglobulin in agglutination, complement fixation (activation), and other antibody reactions and is important in defense against bacteria and viruses. It can be produced by the fetus in certain infections. It has the highest avidity of the immunoglobulins; its interaction with antigen can involve all 10 of its binding sites.


This immunoglobulin has no known antibody function but may function as an antigen receptor; it is present on the surface of many B lymphocytes. It is present in small amounts in serum.


IgE is medically important for two reasons: (1) it mediates immediate (anaphylactic) hypersensitivity (see Chapter 65), and (2) it participates in host defenses against certain parasites (e.g., helminths [worms]) (see Chapter 56). The Fc region of IgE binds to the surface of mast cells and basophils. Bound IgE serves as a receptor for antigen (allergen). When the antigen-binding sites of adjacent IgEs are cross-linked by allergens, several mediators are released by the cells, and immediate (anaphylactic) hypersensitivity reactions occur (see Figure 65–1). Although IgE is present in trace amounts in normal serum (approximately 0.004%), persons with allergic reactivity have greatly increased amounts, and IgE may appear in external secretions. IgE does not fix complement and does not cross the placenta.

IgE is the main host defense against certain important helminth (worm) infections, such as Strongyloides, Trichinella, Ascaris, and the hookworms Necator and Ancylostoma. The serum IgE level is usually increased in these infections. Because worms are too large to be ingested by phagocytes, they are killed by eosinophils that release worm-destroying enzymes. IgE specific for worm proteins binds to receptors on eosinophils, triggering the antibody-dependent cellular cytotoxicity (ADCC) response.


Because immunoglobulins are proteins, they are antigenic, and that property allows them to be subdivided into isotypes, allotypes, and idiotypes.

(1) Isotypes are defined by antigenic (amino acid) differences in their constant regions. Although different antigenically, all isotypes are found in all normal humans. For example, IgG and IgM are different isotypes; the constant region of their H chains (γ and μ) is different antigenically (the five immunoglobulin classes—IgG, IgM, IgA, IgD, and IgE—are different isotypes; their H chains are antigenically different). The IgG isotype is subdivided into four subtypes, IgG1, IgG2, IgG3, and IgG4, based on antigenic differences of their heavy chains. Similarly, IgA1 and IgA2 are different isotypes (the antigenicity of the constant region of their H chains is different), and κ and λ chains are different isotypes (their constant regions also differ antigenically).

(2) Allotypes, on the other hand, are additional antigenic features of immunoglobulins that vary among individuals. They vary because the genes that code for the L and H chains are polymorphic, and individuals can have different alleles. For example, the γH chain contains an allotype called Gm, which is due to a one– or two–amino acid difference that provides a different antigenicity to the molecule. Each individual inherits different allelic genes that code for one or another amino acid at the Gm site.5

(3) Idiotypes are the antigenic determinants formed by the specific amino acids in the hypervariable region.6 Each idiotype is unique for the immunoglobulin produced by a specific clone of antibody-producing cells. Anti-idiotype antibody reacts only with the hypervariable region of the specific immunoglobulin molecule that induced it.


To produce the very large number of different immunoglobulin molecules (estimated to be as many as 100 million) without requiring excessive numbers of genes, special genetic mechanisms (e.g., DNA rearrangement and RNA splicing) are used. The DNA rearrangements are performed by recombinases. Two important genes that encode recombinases are RAG-1 and RAG-2 (recombination-activating genes). Mutations in these genes arrest the development of lymphocytes and result in severe combined immunodeficiency (see page 562).

Each of the four immunoglobulin chains consists of two distinct regions: a variable (V) and a constant (C) region. For each type of immunoglobulin chain (i.e., kappa light chain [κL], lambda light chain [λL], and the five heavy chains [γH, αH, μH, εH, and δH]), there is a separate pool of gene segments located on different chromosomes.7 Each pool contains a set of different V gene segments widely separated from the D (diversity, seen only in H chains), J (joining), and C gene segments (Figure 59–5). In the synthesis of an H chain, for example, a particular V region is translocated to lie close to a D segment, several J segments, and a C region. These genes are transcribed into mRNA, and all but one of the J segments are removed by splicing the RNA. During B-cell differentiation, the first translocation brings a VH gene near a Cμ gene, leading to the formation of IgM as the first antibody produced in a primary response. Note that the J (joining) gene does not encode the J chain found in IgM and IgA. Note also that the DNA of the unused V, D, and J genes is discarded so a particular B cell is committed to making antibody with only one specificity.


FIGURE 59–5 Gene rearrangement to produce a μH chain. The antigen-binding site is formed by randomly choosing one of the VH genes, one of the DH genes, and one of the JH genes. After transcription and RNA splicing, the mRNA is translated to produce an immunoglobulin M (IgM) heavy chain. V, variable regions; D, diversity segments; J, joining segments; C, constant region; IVS, intervening sequence. (Modified and reproduced with permission from Stites DP, Terr A, Parslow T, eds. Basic & Clinical Immunology. 8th ed. Originally published by Appleton & Lange. Copyright 1994 McGraw-Hill.)

The V region of each L chain is encoded by two gene segments (V + J). The V region of each H chain is encoded by three gene segments (V + D + J). These various segments are united into one functional V gene by DNA rearrangement. Each of these assembled V genes is then transcribed with the appropriate C genes and spliced to produce an mRNA that codes for the complete peptide chain. L and H chains are synthesized separately on polysomes and then assembled in the cytoplasm by means of disulfide bonds to form H2L2 units. Finally, an oligosaccharide is added to the constant region of the heavy chain, and the immunoglobulin molecule is released from the cell.

The gene organization mechanism outlined above permits the assembly of a very large number of different molecules. Antibody diversity depends on (1) multiple gene segments, (2) their rearrangement into different sequences, (3) the combining of different L and H chains in the assembly of immunoglobulin molecules, and (4) mutations. A fifth mechanism called junctional diversity applies primarily to the antibody heavy chain. Junctional diversity occurs by the addition of new nucleotides at the splice junctions between the V-D and D-J gene segments.

The diversity of the T-cell antigen receptor is also dependent on the joining of V, D, and J gene segments and the combining of different alpha and beta polypeptide chains. However, unlike antibodies, mutations do not play a significant role in the diversity of the T-cell receptor.

Several lymphoid cancers manifest chromosomal translocations involving the VDJ region and a cellular oncogene. For example, in Burkitt’s lymphoma, the c-myc oncogene on chromosome 8 is translocated to a position adjacent to the VDJ region of a heavy-chain gene. The active promoter of the heavy-chain gene increases transcription of the c-myc oncogene, which predisposes to malignancy.


Initially, all B cells carry IgM specific for an antigen and produce IgM antibody in response to exposure to that antigen. Later, gene rearrangement permits the elaboration of antibodies of the same antigenic specificity but of different immunoglobulin classes (Figure 59–6). Note that the antigenic specificity remains the same for the lifetime of the B cell and plasma cell because the specificity is determined by the variable region genes (V, D, and J genes on the heavy chain and V and J genes on the light chain) no matter which heavy-chain constant region is being utilized.


FIGURE 59–6 Gene rearrangement to produce different immunoglobulin (Ig) classes. IgM is formed first because the μ constant region is closest to the VDJ DNA. Later the μ constant region can be switched with a γ, ε, or α constant region to form the heavy chain of IgG, IgE, or IgA, respectively. Note that the antigenic specificity of the B cell remains the same because the VDJ DNA remains the same. V, variable regions; D, diversity segments; J, joining segments; C, constant regions; S, switch sites. (Modified and reproduced with permission from Stites DP, Terr A, Parslow T, eds. Basic & Clinical Immunology. 8th ed. Originally published by Appleton & Lange. Copyright 1994 McGraw-Hill.)

In class switching, the same assembled VH gene can sequentially associate with different CH genes so that the immunoglobulins produced later (IgG, IgA, or IgE) are specific for the same antigen as the original IgM but have different biologic characteristics. This is illustrated in the “class switch” section of Figure 59–6. A different molecular mechanism is involved in the switching from IgM to IgD. In this case, a single mRNA consisting of VDJ CμCδ is initially transcribed and is then spliced into separate VDJ Cμ and VDJ Cδ mRNAs. Mature B cells can, in this manner, express both IgM and IgD (see Figure 59–6, alternative RNA splicing). Note that once a B cell has “class” switched past a certain H chain gene, it can no longer make that class of H chain because the intervening DNA is excised and discarded. Class switching occurs only with heavy chains; light chains do not undergo class switching. “Switch recombinase” is the enzyme that catalyzes the rearrangement of the VDJ genes during class switching.

The control of class switching is dependent on at least two factors. One is the concentration of various interleukins. For example, interleukin (IL)-4 enhances the production of IgE, whereas IL-5 increases IgA (see Table 58–7). The other is the interaction of the CD40 protein on the B cell with CD40 ligand protein on the helper T cell. In hyper-IgM syndrome, the failure to interact properly results in an inability of the B cell to switch to the production of IgG, IgA, or IgE. Therefore, only IgM is made (see Chapter 68).


A single B cell expresses only one L chain gene (either κ or λ) and one H chain gene. In theory, a B cell could express two sets of immunoglobulin genes, a maternal set and a paternal set. But this is not what happens. Only one set of genes is expressed, either maternal or paternal, and the other set is silent (i.e., it is excluded). This is called allelic exclusion. Each individual contains a mixture of B cells, some expressing the paternal genes and others the maternal ones. The mechanism of this exclusion is unknown.


Antibody can act as an enzyme to catalyze the synthesis of ozone (O3) that has microbicidal activity. Antibody can take the singlet oxygen produced by neutrophils and react it with water to produce hydrogen peroxide and O3. The O3 generated can kill Escherichia coli. The catalytic function of antibodies is independent of their antigen specificity and of the requirement to bind to any antigen. The importance of these observations to our host defenses remains to be determined.


1. It’s time to play “Who am I?” I am the first class of antibody to appear, so my presence indicates an active infection rather than an infection that occurred in the past. I can fix complement, which is an important defense against many bacterial infections. I am found in plasma as a pentamer.

(A) IgA

(B) IgD

(C) IgE

(D) IgG

(E) IgM

2. Regarding IgG, which one of the following is the most accurate?

(A) Each IgG molecule has one antigen binding site.

(B) It is the most important antigen receptor on the surface of neutrophils.

(C) During the primary response, it is made in larger amounts than is IgM.

(D) The ability of IgG to fix complement resides on the constant region of the light chain.

(E) It is the only one of the five immunoglobulins that is transferred from mother to fetus in utero.

3. If a person had a mutation in the gene encoding J (joining) chains, which of the following classes of antibodies could NOT be produced?

(A) IgA and IgM

(B) IgA and IgG

(C) IgG and IgE

(D) IgD and IgE

(E) IgM and IgE

4. Regarding the function of the different classes of antibodies, which one of the following statements is the most accurate?

(A) IgE blocks the binding of viruses to the gut mucosa.

(B) IgA acts as an antigen receptor on the surface of B cells.

(C) IgD is our most important defense against worm parasites, such as hookworms.

(D) IgG can activate the alternative pathway of complement, resulting in the production of C3a that degrades the bacterial cell wall.

(E) There are receptors for the heavy chain of IgG on the surface of neutrophils that mediate a host defense process called opsonization.

5. Regarding the genes that encode antibodies, which one of the following is most accurate?

(A) Hypervariable regions are encoded by the genes of both the light and heavy chains.

(B) The genes for the light and heavy chains are linked on the same chromosome adjacent to the HLA locus.

(C) During the production of IgG, the light and the heavy chains acquire the same antigen binding sites by translocation of the same variable genes.

(D) The gene for the constant region of the gamma heavy chain is first in the sequence of heavy chain genes, and that is why IgG is made in greatest amounts.


1. (E)

2. (E)

3. (A)

4. (E)

5. (A)


Questions on the topics discussed in this chapter can be found in the Immunology section of PART XIII: USMLE (National Board) Practice Questions starting on page 713. Also see PART XIV: USMLE (National Board) Practice Examination starting on page 731.

1Multiple myeloma is a malignant disease characterized by an overproduction of plasma cells (B cells). All the myeloma cells in a patient produce the same type of immunoglobulin molecule, which indicates that all the cells arose from a single progenitor. Excess κ or λ L chains are synthesized and appear as dimers in the urine. These are known as Bence Jones proteins and have the unusual attribute of precipitating at 50°C to 60°C but dissolving when the temperature is raised to the boiling point.

2In humans, the ratio of immunoglobulins containing κ chains to those containing λ chains is approximately 2:1.

3Only IgA and IgM have J chains. Only these immunoglobulins exist as multimers (dimers and pentamers, respectively). The J chain initiates the polymerization process, and the multimers are held together by disulfide bonds between their Fc regions.

4The surface monomer IgM and the serum IgM both have μ heavy chains, but the heavy chain of the surface IgM has a hydrophobic sequence that mediates binding within the cell membrane, whereas the serum IgM does not

5Allotypes related to γH chains are called Gm (an abbreviation of gamma); allotypes related to κL chains are called Inv (an abbreviation of a patient’s name).

6Any one of these antigen determinants is called an idiotope.

7The genes for κL, γL, and the five heavy chains are on chromosomes 2, 22, and 14, respectively.

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