Antibodies are soluble proteins produced by plasma cells. They are normally found in peripheral blood and external body fluids such as saliva, tears, and colostrums. Antibodies neutralize viruses or mark antigens or microbes for destruction by phagocytosis or complement lysis. A number of different synonyms for antibodies exist. When serum is placed in an electrical field, blood proteins migrate at different rates, depending on size and charge. Small-molecular-weight albumins migrate rapidly, whereas globulin fractions migrate more slowly. Antibodies are localized in the slow-migrating gamma (γ) fractions and are termed gammaglobulins. Ultra-centrifugation also can be used to separate immunoglobulins on the basis of size. On the basis of the sedimentation rate (Svedberg units) in a centrifugal field, antibodies are divided into three different molecular weights: (1) the 7S antibody fraction with a molecular weight of 150,000, (2) the 11S fraction with a molecular weight of 300,000, and (3) the 19S fraction with a molecular weight of 900,000.

Antibody Structure

Antibodies have a basic structure which consists of pairs of heavy (H) and light (L) chains. Each heavy chain has a molecular weight of 50,000 Daltons. The weight of light chains is approximately 25,000 Daltons. Disulfide bonds link heavy chains and attach light chains to heavy chains. Heavy and light chains consist of several different globular domains that have constant or variable amino acid sequences (Figure 9-1).

Figure 9-1 The N terminal end of immunoglobulin G (IgG) is characterized by sequence variability (V) in both the heavy and light chains, referred to as the *V_H* and *V_L regions,* respectively. The rest of the molecule has a relatively constant (C) structure. The constant portion of the light chain is termed the *C_L region.* The constant portion of the heavy chain is further divided into three structurally discrete regions: C_H1, C_H2, and C_H3. These globular regions, which are stabilized by intrachain disulfide bonds, are referred to as *domains*. The sites at which the antibody binds antigen are located in the variable domains. The hinge region is a segment of heavy chain between the C_H1 and C_H2 domains. Flexibility in this area permits the two antigen-binding sites to operate independently. There is close pairing of the domains except in the C_H2 region. Carbohydrate moieties are attached to the C_H2 domains. (From Roitt I, Brostoff J, Male D, et al: Immunology, ed 7, Philadelphia, 2006, Mosby.)

Heavy-chain constant domains are called constant regions (C_H1, C_H2, and C_H3). Heavy-chain C_H1 segments are linked to the variable (V_H) domain, which is part of the antigen-combining site. The C_H2 region is the hinge region that ensures antibody flexibility—the larger the hinge region, the more flexible is the antibody (Figure 9-2). Flexibility is essential because epitopes are often widely spaced on protein molecules or microbes. Attachment to cellular receptors is facilitated by the antibody C_H3 region. However, some antibody subpopulations express an additional heavy-chain region (C_H4) that restricts binding to select cells.

Figure 9-2 Flexibility of antibody molecules. The two antigen-binding sites of an immunoglobulin (Ig) monomer can simultaneously bind to two determinants separated by varying distances. A, An immunoglobulin molecule is depicted binding to two widely spaced determinants on a cell surface. B, The same antibody is binding to two determinants that are close together. This flexibility is mainly caused by the hinge regions located between the C_H1 and C_H2 domains, which permit independent movement of antigen-binding sites relative to the rest of the molecule. (From Abbas AK, Lichtman AH, Pillai S: Cellular and molecular immunology, ed 6 [updated edition], Philadelphia, 2010, Saunders.)

Light chains attached to the heavy chains also have constant (C_L) and variable region (V_L) domains. The V_L domain contributes to antigen binding. These 24-kiloDalton (kDal) light chains are covalently linked to heavy chains via disulfide bonds.

Antibody specificity is determined by the association of V regions from both heavy and light chains. In essence, V segments form a three-dimensional pocket that is the mirror image of the antigen that elicited its production (Figure 9-3).

Figure 9-3 Antigens can bind in pockets or grooves or on extended surfaces in the binding sites of antibodies. Schematic representations of the different types of binding sites in a Fab fragment of an antibody are shown: *left*, pocket; *center*, groove; *right*, extended surface.

Within the antigen-binding pocket, small 10-amino-acid hypervariable regions determine specificity at the molecular level. Amino acid sequences that form the three-dimensional antigen pocket are termed complementarity-determining regions (CDRs). Each heavy- and light-chain variable region contains three CDRs.

Affinity and Avidity

Antibody binding is described in terms of affinity and avidity. The binding strength or affinity is the result of the interaction between an antibody and a single antigenic determinant. From a practical perspective, antibody affinity is important in determining the rate at which an infection is terminated. Antibodies with high affinity will tightly bind lower concentrations of microbes and quickly terminate an infection. Low-affinity antibodies are most efficient when large antigen concentrations are present, which means that it takes longer to terminate the infections. Antibody avidity is dependent on the number of antibody-combining sites and the number of epitopes in a single antigen. It is the combined strength of multiple interactions between antibodies and epitopes. Avidity will always be geometrically higher than the affinity.

Antibody Fragments

Porter delineated antibody structure by digesting antibodies with proteolytic papain and pepsin. Papain cleaves the heavy-chain C_H2 domains above the disulfide bonds that connect heavy chains and yields three different fragments. The resultant pieces are identical in structure and consist of constant and variable regions of heavy (V_H and C_H1) and light (V_L and C_L) chains linked by disulfide bonds. These two fragments, each having a molecular weight of 60,000, are called Fab (fragment antigen binding). Each Fab binds a single antigen. The remaining fragment, which consists of C_H2 and C_H3 heavy-chain units, is easily crystallized and is called Fc (fraction crystallized). Later studies showed that the Fc portion of antibodies binds to receptors (FcR) on immunocompetent cells (Figure 9-4).

Figure 9-4 Proteolytic fragments of an immunoglobulin G (IgG) molecule. IgG molecules are cleaved by the enzymes papain **(A)** and pepsin **(B)** at the sites indicated by arrows. Papain digestion allows the separation of two antigen-binding regions (the Fab fragments) from the portion of the IgG molecule that binds to complement and Fc receptors (the Fc fragment). Pepsin generates a single bivalent antigen-binding fragment, F(ab′)₂. (From Abbas AK, Lichtman AH, Pillai S: Cellular and molecular immunology, ed 6 [updated edition], Philadelphia, 2010, Saunders.)

Pepsin cleaves IgG heavy chains at a point below the disulfide bond that links two heavy chains. The resulting fragment consists of two Fab linked together by heavy-chain disulfide bonds. The large-molecular-weight fragments are called F(ab’)₂. Each F(ab’)₂ can bind two antigens. The remainder of the antibody is reduced to small peptide fragments that have no biologic function.

Clinical Usefulness of Fab Fragments

Fab fragments are used in the clinic to treat select drug overdoses and envenomations. For example, Fab fragments are commonly used to treat digoxin overdoses or North American Crotalidae (rattlesnake) envenomations. Fab fragments neutralize the drug or the venom in blood, and the resulting equilibrium shifts away from target cell binding. Since the molecular weight of the antigen–Fab complexes are usually less than 65,000 molecular weight (MW), they pass through the kidneys and are eliminated in urine. Patients usually begin to recover within 30 minutes of receiving a bolus of Fab fragments.