Antibodies and Antigens

Antibodies are proteins that are present on the B-cell membrane and are secreted by plasma cells. Serum antibodies are also sometimes referred to as "immunoglobulins." Antibody functions as the effector of the humoral response by binding to antigen and neutralizing it or facilitating its elimination by 1) cross-linking several antigens, forming clusters that are more readily ingested by phagocytic cells or 2) by binding to antigen on a microorganisms thereby activating the complement system, resulting in lysis of the organism. Antibody can also neutralize toxins or viral particles by coating them, which prevents them form binding to host cells.

An antibody consists of 4 polypeptide chains; 2 identical Heavy chains and 2 identical Light chains which are linked by disulfide bridges. Both light and heavy chains cooperate to form the antigen binding surface. The two antigen binding sites are identical, each formed by the N terminal region of a light chain and the N-terminal region of a heavy chain.

Both light and heavy chains have a variable sequence (Fabs) at their N-terminal ends but a constant sequence at their C terminal ends. Light chains have a constant region about 110 amino acids long and a variable region of the same size. The variable region of the heavy chains (at their N-terminus) is also about 110 amino acids long, but the heavy chain constant region is about 3-4 times longer depending on the class.

The efficiency of antigen binding and cross-linking is greatly increased by a flexible hinge region in the heavy chains of the 3 classes, IgG, IgD and IgA.  Antibodies with the same antigen binding sites can also have any one of several different tail regions (F_c) which gives the antibody different functional properties, such as the ability to activate the complement system, to bind to phagocytic cells or to cross the placenta from mother to fetus. However, the F_c region has no effect on the specificity of the antibody for a particular antigen. Both the hinge region and the tail (F_c) region are formed by the two heavy chains.

There are 5 major classes of heavy chains in humans (alpha, beta, gamma, delta, sigma, and mu) which determine the class or "isotope" of the antibody: Each isotype is distinguished by unique amino acid sequences in the heavy chain constant region that result in structural and functional differences among different isotypes. The 5 antibody classes have different effector functions such as opsonization or the promotion of phagocytosis of antigens by macrophages and neutrophils, antibody dependent cell-mediated cytotoxicity (ADCC) which can kill antibody bound target cells. The various classes are as follows:

• IgG is the most abundant isotype in serum. There are 4 IgG subclasses in humans. The subtle amino acid differences between these subclasses affect biological activity. For example, IgG1, IgG3, and IgG4 readily cross the placenta and protect the developing fetus. IgG molecules are the only antibodies that can pass from mother to fetus in the placenta. Several IgG subclasses are activators of the complement system.

• IgM is the first class produced in a primary response to an antigen. IgM often has a lower affinity than IgG which means that the combined strenght of the noncovalent interactions between a single antigen binding site on IgM and a single epitope is lower for IgM. However, IgM can still bind antigen more effectively than IgG due to its high avidity which is the strenght of multiple interactions between a multivalent antibody and an antigen. IgM is pentameric which means that it has a higher valency (number of binding sites per antibody molecule) than IgG. Thus IgM antiboides typically can bind multiple antigens.

• IgA is the predominant class in external secretions such as breast milk, saliva, tears and mucus of the bronchial, enitourinary and digestive tracts. Secretory IgA is important with defense against certain bacteria like gonorrhea and viruses like polio.

• IgE antibodies mediate the immediate hypersensitivity reactions that are responsible for the symptoms of hay fever, asthma, hives and anaphylactic shock.

• IgD was discovered when a patient developed multiple myelomas whose myelomas protein failed to react with anti-isotype antisera against the then known isotypes. When rabbits were immunized with this myeloma protein, the resulting antisera identified this same class of antibody at low levels in normal human serum. Ig D, with IgM, is the major membrane bound immunoglobulin expressed by mature, B cells and is thought to function in the activation of a B cell by an antigen.

Immunoglobulins are expressed in 2 forms: secreted antibody that is produced by plasma cells, and membrane bound antibody that associates with Ig-α/Ig-Β heterodimers to form the B-cell antigen receptor present on the surface of B cells.  B cells express different isotypes of membrane immunoglobulin ("mIg") at different developmental stages. The immature B cells expressed only mIgM. mIgD appears later in maturation and is the predominant isotype on mature resting B cells. A memory B cell can express a variety of isotypes including combinations of mIgM, mIgG, mIgA and mIgE. Even when different isotypes are expressed on a single cell, the antigenic specificity of all the membrane antibody molecules is the same, so that each antibody molecule binds to the same epitope.

Within a species, each normal individual will express all isotypes in their serum. Different species inherit different constant region genes and therefore express different isotypes. (thus when antibody from one species is injected into another species, the isotypic determinants will be recognized as foreign, inducing an antibody response to the isotype. Such anti isotype antibody is often used to determine the class or subclass of serum antibody produced during an immune response or to characterize the class of membrane bound antibody present on B cells.

Although all members of a species inherit the same set of isotype genes, multiple alleles exist for some of the genes. These alleles encode subtle aa differences, called "allotypic determinants" that occur in some members of a species. Each of these allotypic determinants represents differences in 1-3 aa that are encoded by different alleles. Antibody to allotypic determinants can be produced by injecting antibodies from one member of a species into another who lacks the allotypic determinant.

Sequence variability (generation of immune diversity) of the V_H and V_L chains is concentrated in several hypervariable regions which also form the antigen binding site of the antibody molecule. This antigen binding site is complementary to the structure of the epitope and these regions are sometimes referred to as "complementarity-determining regions (CDRs)." The remaining variable regions are far less variable and sometimes referred to as "framework regions (FRs)." The framework regions of virtually all of the antibodies can be superimposed on one another. Only the CDR loops show different orientations through high resolution xray crystallography. The V_H appears to contribute more to antigen binding than the V_L domain.

The structure of the immunoglobulin molecule is determined by its primary, secondary, tertiary, and quaternary protein structure. The primary aa sequence accounts for the variable and constant regions of the H and L chains. The secondary structure is formed as the chain folds back and forth upon itself forming an antiparallel B sheet which is stabilized by an intrachain disulfide bond and by hydrogen bonds that connect the peptide bonds in neighboring chains. The chains fold into a tertiary structure of compact globular domains. These domains of adjacent H and L chains interact in the quaternary structure to form domains that enable the molecule to specifically bind antigen.

Although Ag-Ab reactions are highly specific, in some cases antibody elicited by one antigen can cross-react with an unrelated antigen where the two antigens share identical or very similar epitopes. This happens, for example, between microbial antigens present on common intestinal bacteria in the gut and red blood cells. Such microbial antigens induce the formation of antibodies in individuals lacking similar blood group antigens (ABO blood group antigens which are glycoproteins expressed on red blood cells) on their red blood cells (In individuals possessing these antigens, complementary antibodies would be eliminated during time that antibodies that recognize self epitopes are negatively selected). The blood group antibodies, although elicited by microbial antigens, will cross-react with similar oligosaccharides on foreign red blood cells. This provides the basis for blood testing. A type A individual has anti-B antibodies; a type B individual has anti-A and a type O individual has anti-A and anti-B. Some vacines also exhibit cross-reactivity. For example, vaccinia virus which causes cowpox expresses cross-reacting epitopes with variola virus which causes smallpox. This fact serves as the basis for using vaccinia virus to induce immunity to smallpox.

Antigens are foreign substances that induce an immune response when injected into an animal. Most large molecules, including virtually all proteins and many polysaccharides can serve as antigens. Those parts of an antigen that combine with the antigen-binding site on either an antibody molecule or a lymphocyte receptor are called antigenic determinants or epitopes. Most antigens have a variety of epitopes that can stimulate the production of antibodies, specific T cell responses, or both. Some epitopes of an antigen produce a greater response than others and are said to be immunodominant.

Most antigens offer multiple epitopes and thus induce proliferation and differentiation of a variety of B-cell clones, each derived form a B cell that recognizes a particular epitope. The resulting serum antibodies are heterogeneous, comprising a mixture of antibodies, each specific for one epitope and are called  polyclonal  as opposed to a single B or T cell clone which is said to be monoclonal

Antibody-Antigen Interactions:

The interaction between an antibody and an antigen depends on 4 types of noncovalent forces:

• ionic bonds between oppositely charged residues;
• hydrogen bonds in which a hydrogen atom is shared between 2 electronegative atoms;
• hydrophobic interactions in which water forces hydrophobic groups together to maximize hydrogen bonding of water molecules, and
• van der Waals interactions between the outer electron clouds of 2 atoms.