Key Notes
Antibodies, often termed ‘immunoglobulins’, are glycoproteins that bind antigens with high specificity and affinity. In humans there are five chemically and physically distinct classes of antibodies (IgG, IgA, IgM, IgD, IgE).
Antibodies have a basic unit of four polypeptide chains – two identical pairs of light (L) chains and heavy (H) chains – bound together by covalent disulfide bridges as well as by noncovalent interactions. These molecules can be proteolytically cleaved to yield two Fab fragments (the antigen-binding part of the molecules) and an Fc fragment (the part of the molecule responsible for effector functions, e.g. complement activation). Both H- and L-chains are divided into V and C regions – the V regions containing the antigen-binding site and the C region determining the fate of the antigen.
The tightness of binding of an antibody-binding site to an antigenic determinant is called its affinity. The tighter the binding, the less likely the antibody is to dissociate from antigen. Different antibodies to an antigenic determinant vary considerably in their affinity for that determinant.
Antibodies produced by a memory response have higher affinity than those in a primary response.
The valence of an antibody is the number of antigenic determinants with which it can react. Having multiple binding sites for an antigen dramatically increases its binding (avidity) to antigens on particles such as bacteria or viruses. For example, two binding sites on IgG are 100 times more effective at neutralizing virus than two unlinked binding sites.
Related topics
Antibodies are glycoproteins that bind antigens with high specificity and affin- ity (they hold on tightly). They are molecules, originally identified in the serum, which are also referred to as ‘immunoglobulins,’ a term often used interchange- ably with antibodies. In humans there are five chemically and physically distinct classes of antibodies (IgG, IgA, IgM, IgD, IgE).
Antibody units All antibodies have the same basic four polypeptide chain unit: two light (L) chains and two heavy (H) chains (Fig. 1). In this basic unit, one L-chain is bound, by a disulfide bridge and noncovalent interactions, to one H-chain.
Similarly, the two H-chains are bound together by covalent disulfide bridges as well as by noncovalent hydrophilic and hydrophobic interactions. There are five different kinds of H-chains (referred to as µ, δ, γ, εand αchains), which deter- mine the class of antibody (IgM, IgD, IgG, IgE and IgA, respectively). There are Molecular
components
Antibody classes (D2) Generation of diversity (D3)
The B cell receptor complex, co-receptors and signaling (E1) Antibody units
Molecular components
Affinity
Antibody valence and avidity
also two different kinds of L-chains –κand λ, each with a MW of 23 kDa. Each antibody unit can have only κ or λL-chains but not both. The properties of the different antibody classes are shown in Table 1.
Both H- and L-chains have intrachain disulfide bridges every 90 amino acid residues, which create polypeptide loops, domains, of 110 amino acids. These domains are referred to as VH, VL, CH1, CH2, etc. (Fig. 1) and have particular functional properties (e.g. VH and VL together form the binding site for anti- gen). This type of structure is characteristic of many other molecules, which are thus said to belong to the immunoglobulin gene superfamily.
The N terminal half of the H-chain and all of the L-chain together make up what is called a Fab fragment (Fig. 1) and contains the antigen-binding site. The actual binding site of the antibody is composed of the N-terminal quarter of the H-chain combined with the N terminal half of the L-chain. The amino acid sequences of these regions differ from one antibody to another and are thus called variable (V) regions and contain the amino acid residues involved in binding an antigenic determinant. Most of the antibody molecule (the C termi- nal three-quarters of the H-chain and the C terminal half of the L-chain) are
62 Section D – Antibodies
CH3CH2
CH1 VH
CL VL
Constant regions Variable regions
Hinge region
Interchain disulfide bonds
Heavy chain hypervariable regions Light chain
Heavy chain Light chain
hypervariable regions
Fab
Fc
Biological activityAntigen binding
Fig. 1. IgG immunoglobulin: basic 4 chain structure representative of all immunoglobulins.
Table 1. Properties of the human immunoglobulins
IgG IgA IgM IgD IgE
Physical properties
Molecular weight, kDa 150 170–420 900 180 190
H-chain MW, kDa 50–55 62 65 70 75
Physiologic properties
Normal adult serum (mg/ml) 8–16 1.4–4.0 0.4–2.0 0.03 ngs
Half-life in days 23 6 5 3 <3
Biologic properties
Complement-fixing capacity + – ++++ – –
Anaphylactic (Type I) hypersensitivity – – – – ++++
Placental transport to fetus + – – – –
There are four IgG (IgG1, IgG2, IgG3, IgG4), two IgA subclasses (IgA1, IgA2) and two L chain types (κand λ).
constant (C) regions of the antibody molecule and are the same for all anti- bodies of the same class and subclass. These C regions do not bind antigen, but rather determine the ‘biological’ properties of the molecule and thus the fate of antigen bound by the antigen-binding site. In particular, the C terminal half of the H-chain, the Fc region (Fragment that crystallized), serves others functions, i.e., combines with complement, is cytophilic (binds to certain types of cells, such as macrophages), etc. Carbohydrates are also present on antibodies, prima- rily on the Fc portion of H-chains.
Affinity Different antibody molecules produced in response to a particular antigenic determinant may vary considerably in their tightness of binding to that deter- minant (i.e., in their affinity for the antigenic determinant). The higher the binding constant the less likely the antibody is to dissociate from the antigen.
Clearly, the affinity of an antibody population is critical when the antigen is a toxin or virus and must be neutralized by rapid and firm combination with antibody. Antibodies formed soon after the injection of an antigen are generally of lower affinity for that antigen whereas antibodies produced later have dramatically greater affinities (association constants 1000 times higher).
The valence of an antibody is the maximum number of antigenic determinants with which it can react. For example, IgG antibodies contain two Fab regions and can bind two molecules of antigen or two identical sites on the same parti- cle, and thus have a valence of two. Valence is important for binding affinity, as having two or more binding sites for an antigen can dramatically increase the tightness of binding of the antibody to antigens on a bacteria or virus. This combined effect, avidity, results from synergy of the binding strengths of each binding site. Avidity is the firmness of association between a multideterminant antigen and the antibodies produced against it.
Determining the avidity of an antibody population is very difficult, since it involves evaluating some function of the group interactions of a large number of different antibodies with a large number of different antigenic determinants.
Even so, the importance of avidity can be demonstrated both mathematically and biologically. For example, as a result of working together (being on the same molecule) two IgG binding sites are 10–100 fold more effective at neutral- izing a virus than two unassociated binding sites, and if the antibody has more binding sites, as in the case of IgM (Topic D2), it may be a million times more effective (Fig. 2). This can be visualized by considering antibodies with one or two binding sites for a particular antigenic determinant on a microorganism.
The antibody with one site can bind to, but can also dissociate from, a determi- nant on the organism. When it comes off, it can diffuse away. However, the antibody with two sites can bind two identical determinants on the organism (each organism has many copies of each protein or carbohydrate). If one bind- ing site dissociates, the other is probably still attached and permits the first site to reform its association with the organism. It therefore follows that the larger the number of binding sites per antibody molecule, the larger the number of bonds formed with an organism, and the less likely it will be to dissociate.
Thus, an antibody with a poor intrinsic affinity for an antigenic determinant can, as a result of a large number of combining sites per molecule, be extremely effective in neutralizing a virus or complexing with a microorganism.
Antibody valence and avidity
D1 – Antibody structure 63
64 Section D – Antibodies
Binding sites 1 2 10
IgM IgG
Fab
Fab IgG IgM
Relative binding avidity 1 100 1 000 000
V ir u s sur f a ce
Fig. 2. Avidity and antibody valence in viral neutralization.
Section D – Antibodies