All of the functions of antibodies are dependent on their ability to specifically bind antigens. We will now consider the nature of antigens and how they are recognized by antibodies.

Features of Biologic Antigens

An antigen is any substance that may be specifically bound by an antibody molecule or T-cell receptor. Antibodies can recognize as antigens almost every kind of biologic molecule, including simple intermediary metabolites, sugars, lipids, autacoids, and hormones, as well as macromolecules such as complex carbohydrates, phospholipids, nucleic acids, and proteins. This is in contrast to T cells, which mainly recognize peptides.

Not all antigens recognized by specific lymphocytes or by secreted antibodies are capable of activating lymphocytes. Molecules that stimulate immune responses are called immunogens. Macromolecules are effective at stimulating B lymphocytes to initiate humoral immune responses because B-cell activation requires bringing together (cross-linking) multiple antigen receptors. Small chemicals, such as dinitrophenol, may bind to antibodies and are therefore antigens, but they cannot activate B cells on their own (i.e., they are not immunogenic). To generate antibodies specific for such small chemicals, immunologists commonly attach multiple copies of the small molecules to a protein or polysaccharide before immunization. In these cases, the small chemical is called a hapten and the large molecule to which it is conjugated is called a carrier. The hapten-carrier complex, unlike free hapten, can act as an immunogen, and this phenomenon has been exploited to produce effective vaccines.

Macromolecules, such as proteins, polysaccharides, and nucleic acids, are usually much bigger than the antigen-binding region of an antibody molecule (see Fig. 1). Therefore any antibody binds to only a portion of the macromolecule, which is called a determinant or an epitope. These two words are syn onymous and are used interchangeably throughout this book. Macromolecules typically contain multiple determinants, some of which may be repeated and each of which, by definition, can be bound by an antibody. The presence of multiple identical determinants in an antigen is referred to as polyvalency or multivalency. Most globular proteins do not contain multiple identical determinants and are not individually polyvalent, but many identical proteins may be displayed in a polyvalent array on cell surfaces, including the surface of microbes. In the case of polysaccharides and nucleic acids, many identical epitopes may be regularly spaced and repeated in the same molecule, and these molecules are said to be polyvalent. Polyvalent arrays of carbohydrate antigens can also be displayed on cell surfaces. Polyvalent antigens can induce clustering of the B-cell receptor and thus initiate the process of B-cell activation.

Fig1. Binding of an antigen by an antibody. (A) A schematic view of complementarity-determining regions (CDRs) generating an antigen-binding site. CDRs from the heavy chain and the light chain are loops that protrude from the surface of the two immunoglobulin V domains and in combination create an antigen binding surface. (B) This model of a globular protein antigen (hen egg lysozyme) bound to an antibody molecule shows how the antigen-binding site can accommodate soluble macromolecules in their native (folded) conformation. The heavy chains of the antibody are red, the light chains are yellow, and the antigen is blue. (C) A view of the interacting surfaces of a hen egg lysozyme (in green) and a Fab fragment of a monoclonal anti hen egg lysozyme antibody (VH in blue and VL in yellow) is provided. The residues of the hen egg lysozyme and of the Fab fragment that interact with each other are shown in red. A critical glutamine residue on lysozyme (in magenta) fits into a “cleft” in the antibody. (B, Courtesy Dr. Dan Vaughn, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. C, From Amit AG, Mariuzza RA, Phillips SE, Poljak RJ. Three dimensional structure of an antigen antibody complex at 2.8 A resolution. Science. 1986;233:747–753.)

The spatial arrangement of different epitopes on a single protein molecule may influence the binding of antibodies in several ways. When determinants are well separated, two or more antibody molecules can be bound to the same protein antigen without influencing each other; such determinants are said to be nonoverlapping. When two determinants are close to each other, the binding of an antibody to the first determinant may cause steric interference with the binding of an antibody to the second; such determinants are said to be overlapping. In rare cases, the binding of one antibody may cause a conformational change in the structure of the antigen, positively or negatively influencing the binding of a second antibody at another site on the protein by means other than steric hindrance. Such interactions are called allosteric effects.

Any available shape or surface on a molecule that may be recognized by an antibody constitutes an antigenic determinant or epitope. Antigenic determinants may be present on carbohydrates, proteins, lipids, and nucleic acids, but also on any other molecule found in nature or generated synthetically. Clinically relevant examples of nonmacromolecular antigens include the catechols found in poison ivy, penicillins, and numerous other small molecules that cause drug allergies. In the case of protein antigens, the formation of some epitopes depends only on the primary structure, and the formation of other determinants reflects tertiary structure, or conformation (shape) (Fig. 2). Epitopes formed by several adjacent amino acid residues are called linear epitopes. The antigen-binding site of an antibody can usually accommodate a linear epitope made up of about six amino acids. If linear epitopes appear on the external surface or in a region of extended conformation in the native folded protein, they may be accessible to antibodies. In other cases, linear epitopes may be inaccessible in the native conformation and appear only when the protein is denatured. In contrast, conformational epitopes are formed by amino acid residues that are not in a sequence but become spatially juxtaposed in the folded protein. Antibodies specific for certain linear epitopes and antibodies specific for conformational epitopes can be used to ascertain whether a protein is denatured or in its native conformation, respectively. Proteins may be subjected to modifications such as glycosylation, phosphorylation, ubiquitination, acetylation, and proteolysis. These modifications, by altering the structure of the protein, can produce new epitopes. New epitopes may also be generated in tumors by alterations in genes encoding self proteins. Such epitopes, induced by posttranslational modifications or by mutation, are called neoantigenic epitopes, and they may be recognized by T lymphocytes and sometimes by specific antibodies.

Fig2. The nature of antigenic determinants. Antigenic determinants (shown in orange, red, and blue) may depend on protein folding (conformation) as well as on primary structure. Some determinants are accessible in native proteins and are lost on denaturation (A), whereas others are exposed only on protein unfolding (B). Neodeterminants arise from postsynthetic modifications, such as peptide bond cleavage (C). Ig, Immunoglobulin.

Structural and Chemical Basis of Antigen

Binding The antigen-binding sites of many antibodies are planar surfaces that can accommodate conformational epitopes of macromolecules, allowing the antibodies to bind large macro molecules (see Fig. 1). The six CDRs, three from the heavy chain and three from the light chain, can spread out to form a broad surface. In a number of antibodies specific for small molecules, such as monosaccharides and drugs, the antigen is bound in a cleft generated by the close apposition of CDRs from the VL and VH domains.

The recognition of antigens by antibodies involves noncovalent, reversible binding. Various types of noncovalent interactions may contribute to antibody binding of antigens, including electro static forces, hydrogen bonds, van der Waals forces, and hydrophobic interactions. The relative importance of each of these depends on the structures of the binding site of the individual antibody and of the antigenic determinant. The strength of the binding between a single combining site of an antibody and an epitope of an anti gen is called the affinity of the antibody. The affinity is commonly represented by the equilibrium dissociation constant (K_d), which indicates how easy it is to separate (dissociate) an antigen-antibody complex into its constituents. The K_d is the ratio of the “off-rate,” a measure of how quickly antigen dissociates from an antibody, to the “on-rate,” which measures how quickly an antigen binds an antibody. A smaller K_d indicates a stronger or higher affinity inter action. The K_d of antibodies produced in typical humoral immune responses usually ranges from about 10⁻⁷ to 10⁻¹¹ M. Serum from an immunized individual will contain a mixture of antibodies with different affinities for the antigen.

Because the hinge region of antibodies gives them flexibility, a single antibody may attach to a single multivalent antigen by more than one binding site. For IgG or IgE, this attachment can involve, at most, two binding sites, one on each Fab. For pentameric IgM, however, a single antibody may bind at up to 10 different sites (Fig. 3). Polyvalent antigens will have more than one copy of a particular determinant. Although the affinity of any one antigen-binding site will be the same for each epitope of a polyvalent antigen, the strength of attachment of the antibody to the antigen must take into account binding of all the sites of the antibody to all the available epitopes of the antigen. This overall strength of attachment is called the avidity and is much greater than the affinity of any one antigen-binding site. Thus, a low-affinity IgM molecule can still bind tightly to a polyvalent antigen because many low-affinity interactions (up to 10 per IgM molecule) can produce a high-avidity interaction.

Fig3. Valency and avidity of antibody-antigen interactions. Monovalent antigens, or epitopes spaced far apart on cell surfaces, will interact with a single binding site of one antibody molecule. Although the affinity of this interaction may be high, the overall avidity may be relatively low. When repeated determinants on a cell surface are close enough, both the antigen-binding sites of a single immunoglobulin G (IgG) molecule can bind, leading to a higher avidity bivalent interaction. The hinge region of the IgG molecule accommodates the shape change needed for simultaneous engagement of both binding sites. IgM molecules have 10 identical antigen-binding sites that can theoretically bind simultaneously with 10 repeating determinants on a cell surface, resulting in a polyvalent, high-avidity interaction.

Polyvalent antigens are important from the viewpoint of B-cell activation, as discussed earlier. Polyvalent interactions between antigens and antibodies are also of biologic significance because many effector functions of antibodies are triggered optimally when two or more antibody molecules are brought close together by binding to a polyvalent antigen. If a polyvalent antigen is mixed with a specific antibody in a test tube, the two interact to form immune complexes (Fig. 4). At the correct concentration, called a zone of equivalence, the antibody and antigen form an extensively cross-linked network of attached molecules such that most or all of the antigen and antibody molecules are complexed into large masses that precipitate out of solution. Immune complexes may be dissociated into smaller aggregates that do not precipitate, either by increasing the concentration of antigen so that free antigen molecules will displace antigens bound to the antibody (zone of antigen excess) or by increasing the antibody so that free antibody molecules will displace bound antibody from antigen determinants (zone of antibody excess). If a zone of equivalence is reached in vivo, large immune complexes can form in the circulation. Immune complexes that are trapped or formed in the walls of blood vessels can initiate an inflammatory reaction, resulting in immune complex diseases.

Fig4. Antigen-antibody complexes. The sizes of antigen-antibody (immune) complexes are a function of the relative concentrations of antigen and antibody. Large complexes are formed at concentrations of multi valent antigens and antibodies that are termed the zone of equivalence; the complexes are smaller in relative antigen or antibody excess.

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مواضيع ذات صلة

NOD-Like Receptors: NOD1 and NOD2

Recognition of Microbes and Damaged Tissue by the Innate Immune System

Overview of Innate Immunity

Transplantation of Tissues and Organs

Rh Blood Types

O-A-B Blood Types

Allergy and Hypersensitivity

The Antiviral Response

Migration of B Lymphocytes

Migration of Effector T Lymphocytes to Sites of Infection

Exit of Naive and Effector T Cells from Lymph Nodes

Lymphocytes are Responsible for Acquired Immunity