An antibody is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target.
Immunoglobulins (Ig) are glycoproteins that function as antibodies. They are found in the blood and tissue fluids, as well as many secretions. Structurally they are globulins (in the γ-region of protein electrophoresis). They are synthesized and secreted by B cells of the immune system. B cells are activated upon binding to their specific antigen. In some cases the interaction of the B cell with a T helper cell is also necessary.
Structure of the antibody
Immunoglobulins are heavy plasma proteins, often with added sugar chains (see glycosylation) on N-terminal (all antibodies) and occasionally O-terminal (IgA1 and IgD) aminoacid residues.
The basic unit of each antibody is a monomer. An antibody can be monomeric, dimeric, trimeric, tetrameric, pentameric etc. The monomer is a "Y"-shaped molecule that consists of two identical heavy chains and two identical light chains connected by disulfide bonds.
There are five types of heavy chain: γ, δ, α, μ and ε. They define classes of immunoglobulins. Heavy chains α and γ have approximately 450 amino acids, while μ and ε have approximately 550 amino acids. Each heavy chain has a constant region, which is the same by all immunoglobulins of the same class, and a variable region which differs between immunoglobulins of different B cells, but is the same by all immunoglobulins of the same B cell. Heavy chains γ, α and δ have the constant region composed of three domains; the constant region of heavy chains μ and ε is composed of four domains. The variable domain of any heavy chain is composed of one domain. These domains are about 110 amino acids long. There are also some amino acids between constant domains.
There are only two types of light chain: λ and κ. In humans they are similar, but only one type is present in each antibody. Each light chain has two successive domains: one constant and one variable domain. The approximate length of a light chain is from 211 to 217 amino acids.
The monomer is composed of two heavy and two light chains. Together this gives six to eight constant domains and four variable domains. If it is cleaved with enzymes papain and pepsin, we get two Fab (fragment binding antigen) fragments and an Fc (fragment crystallizable) fragment.
Each half of the forked end of the "Y" shaped monomer is called the Fab fragment. It is composed of one constant and one variable domain of each the heavy and the light chain, which together shape the antigen binding site at the amino terminal end of the monomer. The two variable domains bind the antigens they are specific for and that elicited their production.
The ability to bind a wide variety of foreign antigens arises from events know as somatic recombination. This is when genes are selected (variable (V), diversity (D) and joining (J) for heavy chains, and only V and J for light chains) to form countless combinations. The main reason that the human immune system is capable of binding so many antigens is the variable region of the heavy chain. More specifically, it is the area where these V, D and J genes are found - otherwise known as the complementarity determining region 3 (CDR3).
The Fc fragment is the stem of the "Y" and is composed from two heavy chains that each contribute two to three constant domains (depending on the class of the antibody). It binds to various cell receptors and complement proteins. In this way it mediates different physiological effects of antibodies (opsonization, cell lysis, mast cell, basophil and eosinophil degranulation and other processes).
The variable regions of the heavy and light chains can be fused together to form a single chain variable fragment (scFv), which retains the original specificity of the parent immunoglobulin.
A crude estimation of immunoglobulin levels can be made by protein electrophoresis. Here the plasma proteins are separated into albumin, alpha-globulins (1 and 2), beta-globulins (1 and 2) and gamma-globulins according to weight. Immunoglobulins are all in the gamma region. In some disease states (myeloma) a very high concentration of one particular immunoglobulin will show up as a monoclonal band.
According to differences in their heavy chain constant domains, immunoglobulins are grouped into five classes or isotypes: IgG, IgA, IgM, IgD, and IgE. (The isotypes are also defined with light chains, but they do not define classes, so they are often neglected.) Other immune cells partner with antibodies to eliminate pathogens depending on which IgG, IgA, IgM, IgD, and IgE constant binding domain receptors it can express on its surface.
The antibodies a single B lymphocyte produces can differ in their heavy chain and the B cell often expresses different classes of antibodies at the same time. However, they are identical in their specificity for antigen, conferred by their variable region. To achieve the large number of specificities the body needs to protect itself against many different foreign antigens, it must produce millions of B lymphoyctes. It is important to note that to produce such a diversity of antigen binding sites with a separate gene for each possible antigen, the immune system would require many more genes than exist in the genome. Instead, as Susumu Tonegawa showed in 1976, portions of the genome in B lymphocytes can recombine to form all the variation seen in the antibodies and more. Tonegawa won the Nobel Prize in Physiology or Medicine in 1987 for his discovery.
IgG is a monomeric immunoglobulin, built of two heavy chains γ and two light chains. Each molecule has two antigen binding sites. This is the most abundant immunoglobulin and is approximately equally distributed in blood and in tissue liquids. It is important that this is the only isotype that can traverse trough the placenta, because this gives protection to the fetus in its first weeks of life, when its own immune system has not yet developed. It can bind to many kinds of pathogens, for example viruses, bacteria, and fungi and protects the body against them by complement activation (classic pathway), opsonization for phagocytosis and neutralisation (immunology) of their toxins. There are 4 subclasses: IgG1 (66%), IgG2 (23%), IgG3 (7%) and IgG4 (4%).
IgA represent about 15 to 20% of immunoglobulins in the blood although it is primarily secreted across the mucosal tract into the stomach and intestines. It is also found in maternal milk, tears and saliva. This immunoglobulin helps to fight against pathogens that contact the body surface, ingested, or inhaled. It does not activate complement and opsonises only weakly. Its heavy chains are of the type α. It exists in two forms, IgA1 (90%) and IgA2 (10%) that differ in the structure. IgA1 is composed like other proteins, however in IgA2 the heavy and light chains are not linked with disulfide but with noncovalent bonds.
The IgA found in secretions have a special form. They are dimeric molecules, linked by two additional chains. One of these is the J chain (from join), which is a polypeptide of molecular mass 1,5 kD, rich with cysteine and structurally completely different from other immunoglobulin chains. This chain is formed in the antibodies secreting cells. The dimeric form of IgA in the outer secretions has also a polypeptide of the same molecular mass (1,5 kD) that is called the secretory chain and is produced by the epithelial cells. It is also possible to find trimeric and even tetrameric IgA.
IgM forms polymers where multiple immunoglobulins are covalently linked together with disulfide bonds, usually as a pentamer or a hexamer. It has a large molecular mass of approximately 900 kD. The J chain is attached to most pentamers, while hexamers do not possess the J chain due to space constraints in the complex. Because each monomer has two antigen binding sites, an IgM has 10 of them, however it cannot bind 10 antigens at the same time because they hinder each other. Because of its large molecule, it cannot diffuse well, so it is found in the interstitium only in very low quantities. IgM is primarly found in serum, however of the J chain it is also important as a secretory immunoglobulin. Due to its polymeric nature, IgM possesses high avidity, and is particularly effective at complement activation. It is also a so-called "natural antibody". This means that it is found in the serum without an evident contact with antybody.
IgD makes up about 1% in the plasma membranes in B-lymphocytes. It is monomeric with the δ heavy chain. These immunoglobulins are probably involved in the development of differentiated B-lymphocytes into plasma and memory cells.
IgE is a monomeric immunoglobulin with the heavy chain ε. It contains a lot of carbon hydrates. Its molecular mass is 190 kD. It can be found on the surface of the plasma membrane of basophils and mast cells of connective tissue. IgE plays a role in immediate hypersensitivity and the defense of parasites such as worms. The IgE antibodies are present also in outer excretions. They do not activate complement.
The antibodies have two primary functions:
- they bind antigens -- see below
- they combine with different immunoglobulin receptors specific for them and exert effector functions. These receptors are isotype specific, which gives a great flexibility to the immune system, because this enables that in different situations only certain immune mechanisms respond to antigens.
The humoral immune response
When a macrophage ingests a pathogen, it attaches parts of its proteins to a class II MHC protein. This complex is moved to the outside of the cell membrane, where it can be recognized by a T lymphocyte, which compares it to similar structures on the cell membrane of a B lymphocyte. If it finds a matching pair, the T lymphocyte activates the B lymphocyte, which starts producing antibodies. A B lymphocyte can only produce antibodies against the structure it presents on its surface.
Antibodies exist freely in the bloodstream or bound to cell membranes. They are part of the humoral immune system. Antibodies exist in clonal lines that are specific to only one antigen, e.g., a virus hull protein. In binding to such antigens, they can cause agglutination and precipitation of antibody-antigen products prime for phagocytosis by macrophages and other cells, block viral receptors and stimulate other immune responses such as the complement pathway.
Antibodies that recognize viruses can block these directly by their sheer size. The virus will be unable to dock to a cell and infect it, hindered by the antibody. They can also agglutinate them so the phagocytes can capture them.
Antibodies that recognize bacteria mark them for ingestion by macrophages. Together with the plasma component complement, antibodies can kill bacteria directly. They neutralize toxins by binding with them.
It is important to note that antibodies cannot attack pathogens within cells, and certain viruses "hide" inside cells for long periods of time to avoid them. This is the reason for the chronic nature of many minor skin diseases (such as cold sores); any given outbreak is quickly suppressed by the immune system, but the infection is never truly eradicated because some cells retain viruses that will resume it later.
Detection of particular antibodies is a very common form of medical diagnostics. Serology depends on these menthods. Autoimmune disorders can often be traced to antibodies that bind the body's own epitopes; many can be detected through blood tests.
"Designed" monoclonal antibody therapy is already being employed in a number of diseases (including rheumatoid arthritis) and in some forms of cancer. Presently, many antibody-related therapies are undergoing extensive clinical trials for use in practice.
In biochemistry, antibodies are used for immunological identification of proteins, using the Western blot method. A similar technique is used in ELISPOT and ELISA assays, in which detection antibodies are used to detect cell secretions such as cytokines or antibodies. Antibodies are also used to separate proteins (and anything bound to them) from the other molecules in a cell lysate.
These purified antibodies are often produced by injecting the antigen into a small mammal, such as a mouse or rabbit. Blood isolated from these animals contains polyclonal antibodies -- multiple antibodies that stick to the same antigen. The serum (=blood from which blood-clotting proteins and red-blood cells were removed), also known as the antiserum, because it now contains the desired antibodies, is commonly purified with Protein A/G purification or antigen affinity chromatography. If the lymphocytes that produce the antibodies can be isolated and immortalized, then a monoclonal antibody can be obtained. Monoclonal antibodies have much greater specificity than polyclonal antibodies.
- Rhoades, Rodney and Richard Pflanzer (2002). Human Physiology (4th ed.). Brooks/Cole. ISBN 0534421741