All antibody isotypes except IgD are bifunctional

Antibodies are bifunctional molecules. They:

One part of the antibody molecule determines its antigen specificity while another determines which effector functions will be activated. Effector functions include binding of the heavy chain constant regions (Fc) to:

Fig. 3.2 Antibodies act as adapter molecules for immune effector systems

Antibodies act as adapter molecules for different immune effector systems, linking antigens to receptor molecules (C1q and FcR) of the immune system. (1) Immune complexes can activate the complement classical pathway. (2) Antibodies bound to the surface of pathogens opsonize them for phagocytosis. (3) Antibodies bound to cells can promote their recognition and killing by NK cells. (Similarly recognition of some parasitic worms by eosinophils, mediated by antibodies, targets them for killing.) (4) Antibody bound to Fc receptors sensitizes cells so that they can recognize antigen, and the cell becomes activated if antigen binds to the surface antibody.

Antibody class and subclass is determined by the structure of the heavy chain

The basic structure of each antibody molecule is a unit consisting of:

In an individual antibody molecule the amino acid sequences of the two light chains are identical, as are the sequences of the two heavy chains. Both light and both heavy chains are folded into a series of discrete domains. The sequence of the constant region of the heavy chain determines the class and subclass, or isotype, of the antibody. The heavy chains are designated:

There are no subclasses of IgM, IgD, or IgE (Fig. 3.3).

Fig. 3.3 Physicochemical properties of human immunoglobulin classes

Each immunoglobulin class has a characteristic type of heavy chain. Thus IgG possesses γ chains; IgM, μ chains; IgA, α chains; IgD, δ chains; and IgE, ε chains. Variation in heavy chain structure within a class gives rise to immunoglobulin subclasses. For example, the human IgG pool consists of four subclasses reflecting four distinct types of heavy chain. The properties of the immunoglobulins vary between the different classes. In secretions, IgA occurs in a dimeric form (sIgA) in association with a protein chain termed the secretory component. The serum concentration of sIgA is very low, whereas the level in intestinal secretions can be very high.

IgG is the predominant antibody isotype in normal human serum

IgG accounts for 70–75% of the total serum antibody pool and consists of a monomeric four-chain molecule. Normal concentration range: 6.0–16 g/L.

IgM accounts for about 10% of the serum antibody pool

IgM accounts for approximately 10% of the serum antibody pool. It is a pentamer of a basic four-chain structure with a mass of ~970 kDa; polymerization to the pentamer is aided by the presence of the J (joining) polypeptide chain of mass ~15 kDa. Normal concentration range: 0.5–2.0 g/L. A transmembrane monomeric form (mIgM) is present as an antigen-specific receptor on mature B cells.

IgA is the predominant antibody isotype present in seromucous secretions

IgA accounts for approximately 15–20% of the serum anibody pool. In humans over 80% of serum IgA has a four chain monomer. Normal concentration range: 0.8–4.0 g/L. In the serum of most mammals IgA is polymeric, mostly as a dimer.

IgA is the predominant antibody isotype present in seromucous secretions such as saliva, colostrum, milk, and tracheobronchial and genitourinary secretions.

IgD is an antigen-specific receptor (mIgD) on mature B cells

IgD, a four chain monomer, accounts for less than 1% of the serum antibody pool. Normal concentration range: 2.0–100 mg/L.

Basophils and mast cells are continuously saturated with IgE

Serum IgE levels are very low (0–90 IU/mL) relative to the other antibody isotypes. However, basophils and mast cells express an IgE-specific receptor of very high affinity with the result that they are continuously saturated with IgE.

Assembled IgM molecules have a ‘star’ conformation

IgM is present in human serum as a pentamer of the basic four-chain structure (see Fig. 3.w3). Each heavy chain is comprised of a VH and four CH domains. One advantage of this pentameric structure is that it provides 10 identical binding sites, which can dramatically increase the avidity with which IgM binds its cognate antigen. Given that serum IgM commonly functions to eliminate bacteria containing low affinity, polysaccharide antigens, the increased avidity provided by the pentameric structure provides an important functional advantage.

Fig. 3.w3 Structural characteristics of various human immunoglobulins

Carbohydrate side chains are shown in blue. Inter heavy (H) chain disulfide bonds are shown in red, but interchain bonds between H and L chains are omitted. (1) A model of IgG1 indicating the globular domains of H and L chains. Note the apposition of the CH3 domains and the separation of the CH2 domains. The carbohydrate units lie between the CH2 domains. (2) Polypeptide chain structure of human IgG3. Note the elongated hinge region. (3) IgM H chains have five domains with disulfide bonds cross-linking adjacent CH3 and CH4 domains. The possible location of the J chain is shown. IgM does not have extended hinge regions, but flexion can occur about the CH2 domains. (4) The secretory component of sIgA is probably wound around the dimer and attached by two disulfide bonds to the CH2 domain of one IgA monomer. The J chain is required to join the two subunits. (5) This diagram of IgD shows the domain structure and a characteristically large number of oligosaccharide units. Note also the presence of a hinge region and short octapeptide tailpieces. (6) IgE can be cleaved by enzymes to give the fragments F(ab′)₂, Fc, and Fc′. Note the absence of a hinge region.

Covalent disulfide bonds between adjacent CH2 and CH3 domains, the C terminal 18-residue peptide sequence, referred to as the ‘tailpiece’, and J chain link the subunits of the pentamer.

J chain is synthesized within plasma cells, has a mass of ~15 kDa and folds to form an immunoglobulin domain. Each heavy chain bears four N-linked oligosaccharide moieties, however, the oligosaccharides are not integral to the protein structure in the same way as in IgG-Fc. Oligosaccharides present on IgM activate the complement cascade via binding to the mannose binding lectin (see Chapter 4).

In electron micrographs the assembled IgM molecule is seen to have a ‘star’ conformation with a densely packed central region and radiating arms (Fig. 3.6); however, electron micrographs of IgM antibodies binding to poliovirus show molecules adopting a ‘staple’ or ‘crab-like’ configuration (see Fig. 3.6), which suggests that flexion readily occurs between the CH2 and CH3 domains, though this region is not structurally homologous to the IgG hinge. Distortion of this region, referred to as dislocation, results in the ‘staple’ configuration of IgM required to activate complement.

Fig. 3.6 Electron micrographs of IgM molecules

(1) In free solution, deer IgM adopts the characteristic star-shaped configuration. ×195 000. (2) Rabbit IgM antibody (arrow) in ‘crab-like’ configuration with partly visible central ring structure bound to a poliovirus virion. × 190 000.

((1) Courtesy of Drs E Holm Nielson, P Storgaard, and Professor S-E Svehag. (2) Courtesy of Dr B Chesebro and Professor S-E Svehag.)

Secretory IgA is a complex of IgA, J chain and secretory component

IgA present in serum is produced by bone marrow plasma cells and secreted as a monomer with the basic four-chain structure. Each heavy chain is comprised of a VH and three CH domains.

The IgA1 and IgA2 subclasses differ substantially in the structure of their hinge regions:

A deficit in the addition of O-linked sugars within the hinge region of IgA1 protein has been linked with the disease IgA nephropathy.

IgA is the predominant antibody isotype in external secretions but is present as a complex secretory form. IgA is secreted by gut localized plasma cells as a dimer in which the heavy chain ‘tailpiece’ is covalently bound to a J chain, through a disulphide bond (see Fig. 3.w3).

Electron micrographs of IgA dimers show double Y-shaped structures, suggesting that the monomeric subunits are linked end-to-end through the C terminal Cα3 regions (Fig. 3.7).

Fig. 3.7 Electron micrographs of human dimeric IgA molecules

The double Y-shaped appearance suggests that the monomeric subunits are linked end to end through the C terminal Cα3 domain. × 250 000.

(Courtesy of Professor S-E Svehag.)

The dimeric form of IgA binds a poly-Ig receptor (Fig. 3.8) expressed on the basolateral surface of epithelial cells. The complex is internalized, transported to the apical surface where the poly-Ig receptor is cleaved to yield the secretory component (SC) that is released still bound to the IgA dimer. The released secretory form of IgA is relatively resistant to cleavage by enzymes in the gut and is comprised of:

Fig. 3.8 Transport of IgA across the mucosal epithelium

IgA dimers secreted into the intestinal lamina propria by plasma cells bind to poly-Ig receptors on the internal (basolateral) surface of the epithelial cells. The sIgA–receptor complex is then endocytosed and transported across the cell while still bound to the membrane of transport vesicles. These vesicles fuse with the plasma membrane at the luminal surface, releasing IgA dimers with bound secretory component derived from cleavage of the receptor. The dimeric IgA is protected from proteolytic enzymes in the lumen by the presence of this secretory component.

Serum IgD has antigen specificity but not effector functions

Serum IgD accounts for less than 1% of the total serum immunoglobulin. Although serum IgD has been shown to have specific antigen binding activity no effector functions have been identified. Each heavy chain is comprised of a VH domain and three CH domains with an extended hinge region (see Figs. 3.w3). IgD also functions as an antigen specific receptor on B cells, together with IgM, and as such exhibits the same diversity of antigen specificity.

The heavy chain of IgE is comprised of four constant region domains

It is estimated that ~50% of total body IgE is present in the blood, with the remainder being bound to mast cells and basophils through their high-affinity IgE Fcε receptor (FcεRI). IgE also binds the low affinity receptor (FcεRII) expressed on B cells and cells of the myeloid lineage.

Each heavy chain is comprised of a VH and four CH domains and bears six N-linked oligosaccharides (see Figs. 3.w3). An N-linked oligosaccharide, present in the CH3 domain, and equivalent to CH2 in IgG, influences binding to FcεRII but not FcεRI.

Antibody isotypes are the products of genes present within the genome of all healthy members of a species

The human immunoglobulin heavy chain isotypes are encoded by μ, δ, γ3, γ1, α1, γ2, γ4, α2, and ε genes on chromosome 14. The single κ and multiple λ isotypes are encoded by Cκ and Cλ genes on chromosomes 2 and 22, respectively.

Allotypes result from genetic variation at a locus within the species

Immunoglobulin allotypes refer to proteins that are the product of polymorphic immunoglobulin genes (i.e. the result of genetic variation at a given locus within the species). The WHO-adopted nomenclature – for example G3m(g) – requires:

The sequence correlates have been determined. For example G1m(f) and G1m(z) allotypes are characterized by the presence of, respectively, an arginine or lysine residue at position 214 in the CH1 or Cγ1 domain of the heavy chains.

In humans allotypes are encountered within:

Because all heavy chain genes are encoded within the same locus, they are inherited as a complex or haplotype. Studies of immunoglobulin haplotypes help in the identification of different population groups of humans.

Allotypes of human IgG were first defined serologically, with antisera obtained from multi-parous women and individuals receiving multiple transfusions. In each case an allotype mis-match resulted in anti-allotype responses.

Idiotypes result from antigenic uniqueness

The structural uniqueness of antibody variable regions can be reflected in antigenic uniqueness recognized by antisera. However, in addition to antigenic uniqueness, cross-reactivity may be observed for two V regions that are highly homologous. The terms private and public (or cross-reactive and recurrent) idiotypes are used to describe this property. Idiotypes:

Idiotypy (from the Greek ‘idios’, meaning ‘private’) originally referred to the antigenic uniqueness of an individual antibody molecule as recognized by antisera raised in rabbits and mice, by immunization of an individual with a single antibody molecule raised in another member of the same species and allotype. For human IgG proteins heterologous antisera are raised, in rabbits or mice, and absorbed with polyclonal IgG, to absorb cross-reactive antibody. In the modern era monoclonal anti-idiotype antibodies would be generated. They are important reagents when generating assays for quantitative and qualitative analysis of an antibody therapeutic (see Method box 3.1).