A European Informational Website
learn more
An antibody or immunoglobulin is a large Y-shaped glycoprotein belonging to the immunoglobulin superfamily that is used by the immune system to identify and neutralize foreign objects like bacteria and viruses. An antibody contains two sites called that recognize a specific target, which is called an antigen.[1] Paratopes can be thought of as similar to locks and are specific for just one particular part of the antigen called an epitope, which can be considered similar to a key. This specific lock and key interaction allows an antibody to tag a microbe or an infected cell for attack by other parts of the immune system. The binding of an antibody can also neutralize its antigen target directly by, for example, blocking a part of a microbe that is essential for its survival and growth in the body.[2] The production of antibodies is the main function of the humoral immune system.[3]
Antibodies occur in two forms: a soluble form that is secreted from cells and released into the blood and tissue fluids, and a membrane-bound form that is attached to the surface of a B cell and is called the B cell receptor (BCR). The BCR on the surface of B cells allows the B cell to detect when a specific antigen is present in the body. Once the B cell binds to an antigen the B cell can be activated - interaction of the B cell with a T helper cell is necessary to produce full activation of the B cell.[4] The activated B cell differentiates into either soluble antibody generating factories called plasma cells, or into memory cells that will survive in the body for years afterwards, allowing an organism to remember that antigen and respond faster upon future exposures.[5]
In mammals there are five antibody isotypes or classes, each named with an "Ig" prefix that stands for immunoglobulin, and known as IgM, IgD, IgG, IgA and IgE. These immunoglobulin classes differ in their biological properties, functional locations and their ability to deal with different antigens, as depicted in the table below.[6] The antibody isotype changes during development and with activation of B cells. Immature B cells that have never been exposed to antigen are known as naïve B cells, and express only cell surface bound IgM. These cells begin to express both IgM and IgD when reaching maturity - the co-expression of both these immunoglobulin isotypes renders the B cell 'mature' and ready to respond to antigen.[7] B cell activation follows engagement of the cell bound immunoglobulin molecule with an antigen, causing the cell to divide and differentiate into an antibody producing cell called a plasma cell. In this activated form, the B cell starts to produce immunoglobulin in a secreted form rather than a membrane-bound form. Some daughter cells of the activated B cells undergo isotype switching, a mechanism that induces production of the other antibody isotypes such as IgE, IgA or, more commonly, IgG, that have defined roles in the immune system.
Immunoglobulins are heavy plasma proteins, with sugar chains added to amino acid residues by N-linked glycosylation and occasionally (e.g. IgA1 and IgD) by O-linked glycosylation.[8] In other words, antibodies are glycoproteins. The basic unit of each antibody is a monomer with one Ig unit but secreted antibody can also be dimeric with two Ig units as with IgA, tetrameric with four Ig units like teleost fish IgM, or pentameric with five Ig units, like mammalian IgM.[9]
The Ig monomer is a "Y"-shaped molecule that consists of four polypeptide chains; two identical heavy chains and two identical light chains connected by disulfide bonds.[6]
There are five types of mammalian immunoglobulin heavy chain denoted by the Greek letters: γ, δ, α, μ and ε.[1] They define the classes of immunoglobulins and correspond to IgG, IgD, IgA, IgM and IgE antibodies, respectively.[2] Heavy chains α and γ have approximately 450 amino acids, while μ and ε have approximately 550 amino acids.[1]
Each heavy chain has two regions, known as the constant region and the variable region. The constant region is the same for all immunoglobulins within the same class, but differs between antibodies from different classes. Heavy chains γ , α and δ have a constant region composed of three tandem (in a line next to each other) immunoglobulin domains, and a hinge region for added flexibility,[6] but heavy chains μ and ε have a constant region composed of four immunoglobulin domains.[1] The variable region of the heavy chain is different for each individual B cell, but is the same for all immunoglobulins produced by the same B cell or B cell clone. The variable domain of any heavy chain is composed of a single immunoglobulin domain. These domains are each about 110 amino acids long.
There are only two types of light chain in mammals, which are called lambda (λ) and kappa (κ).[1] Other types of light chains, such as the iota (ι) chain, are found in lower vertebrates like Chondrichthyes and Teleostei. Each antibody contains two light chains that are always identical; only one type of light chain, κ or λ, is present per antibody in mammals. A light chain has two successive domains: one constant domain and one variable domain. The approximate length of a light chain is 211 to 217 amino acids.[1]
Each end of an antibody has a different function. The tip of the Y contains the paratope and is important for binding antigen and, therefore, for recognizing specific foreign objects. This region is called the Fab (fragment, antigen binding) region of the antibody and is composed of one constant and one variable domain from each heavy and light chain of the antibody.[10] The paratope is shaped at the amino terminal end of the antibody monomer by the variable domains from the heavy and light chains.
The base of the Y is important for binding to specific proteins that modulate immune cell activity, and generates an appropriate immune response for a given antigen through this interaction.[11] This region is called the Fc (Fragment, crystallizable) region, and is composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody.[1] The Fc region binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiological effects including opsonization, cell lysis, and degranulation of mast cells, basophils and eosinophils.[6][12]
Antibody light and heavy chains are composed of structural domains called immunoglobulin (Ig) domains. These domains contain about 70-110 amino acids and are classified into different categories (for example, variable or IgV, and constant or IgC) according to their size and function.[13] They possess a characteristic immunoglobulin fold in which two beta sheets create a “sandwich” shape, held together by interactions between conserved cysteines and other charged amino acids. Although antibodies were the first molecules in which these domains were identified, Ig-like domains are found in all proteins of the immunoglobulin superfamily (IgSF).
The success of antibodies to recognize and eradicate many different types of microbe requires their wide diversity; antibodies vary from one another in their amino acid composition allowing them to interact with different antigens.[14] An individual vertebrate possesses a large number of different antibodies, each capable of binding to a distinct antigen or epitope from a foreign object. However, although a huge number of different antibodies are generated, there is not an equally large array of genes available, in a single individual, to make such a huge repertoire of antibodies. Vertebrate B cells have several mechanisms that allow them to generate large antibody diversity from a relatively small number of immunoglobulin genes.[15]
Somatic recombination of immunoglobulins, also known as V(D)J recombination, involves the random selection and combining of genes encoding each segment of the immunoglobulin variable region in a manner that generates a huge repertoire of antibodies with different . These segments are called variable (V), diversity (D) and joining (J) segments.[15] V, D and J segments are found in Ig heavy chains but only the V and J segments are found in Ig light chains. Multiple copies of the V, D and J segments exist tandemly arranged in the genomes of mammals. Their selection for recombination within the individual B cell is also called gene rearrangement.[16] A B cell that successfully produces a functional immunoglobulin gene during its V(D)J recombination will suppress the expression of any other variable region gene by a process known as allelic exclusion.[17] Thus, the variable regions of all the immunoglobulin molecules within one given B cell will be the same, although the constant domains of the heavy chains can differ.[1] The diversity generated by this mechanism in the variable region of the heavy chain - to be specific, in the area that these V, D and J genes encode, otherwise known as the complementarity determining region 3 (CDR3) - provides the vertebrate immune system its ability to bind so many distinct antigens.
For more details on this topic, see Somatic hypermutation and Affinity maturation
A further mechanism for generating antibody diversity exists for the mature B cell after antigen stimulation. When B cells become activated with an antigen they begin to proliferate. Proliferating B cells produce high levels of point mutations in the genes encoding the variable domains of their antibody heavy and light chains. This is called somatic hypermutation, and results in approximately one nucleotide change, in the immunoglobulin variable genes, per cell division.[18] As a consequence, slight changes occur in the amino acid sequence of the variable domains of both the light and heavy chains between clones of the same activated B cell, and ultimately, differences in the affinity or strength of interaction that the B cell has with its specific antigen.[19] Some mutations generate antibodies that have a weaker interaction (low affinity) with their antigen than the original antibody, and some generate antibodies with a stronger interaction (high affinity).[20] The B cells use their cell-bound antibodies to interact with antibody-antigen complexes bound by Fc receptors on other immune cells called follicular dendritic cells; this interaction is stabilized by a secondary interaction between the antibody and complement bound antigen/complement receptor complexes. B cells that express high affinity antibodies on their surface will receive a strong survival signal from this interaction, whereas those with low affinity antibodies will not, and will die by apoptosis to leave only the cells that generate higher affinity antibodies.[20] Thus, B cells expressing immunoglobulins with higher affinity for the antigen will outcompete those with weaker immunoglobulin for function and survival. This process of generating antibodies with higher binding affinities for a given antigen than the original binding antibody is called affinity maturation, occurs in mature B cells after V(D)J recombination, and is dependent on help from helper T cells.[21]
Isotype switching (or class switching) occurs after the process of V(D)J recombination and following activation of the mature B cell (see above) to generate the different classes of antibody, all with the same variable domains as the original immunoglobulin generated in the immature B cell during recombination, but possessing distinct constant domains in their heavy chains.[16]
Naïve B cells produce both IgM and IgD that have identical antigen binding regions. After activation by antigen, some B cells undergo a biological process known as class switching, which allows them to produce antibodies of the IgG, IgA or IgE classes. During class switching, the constant region of the immunoglobulin heavy chain changes but the variable regions, and therefore antigen specificity, stay the same. This allows different daughter cells from the same activated B cell to produce antibodies of different isotypes.[22]
Class switching occurs by a mechanism called class switch recombination (CSR). This process uses conserved nucleotide motifs, called switch (S) regions, found in DNA upstream from each of the antibody heavy chain constant region genes, except the δ-chain. DNA is nicked and broken at two selected S-regions by the activity of a series of enzymes, including Activation-Induced (Cytidine) Deaminase (AID), uracil DNA glycosylase and apyrimidic/apurinic (AP)-endonucleases.[23][24] The intervening DNA between the S-regions is subsequently deleted from the chromosome, removing unwanted μ or δ heavy chain constant region genes and allowing substitution of the γ, α or ε constant region genes. The free ends of the DNA are rejoined by a process called non-homologous end joining (NHEJ) to link the variable domain exon to the desired downstream constant domain exon of the immunoglobulin heavy chain.[25]
Antibodies reversibly bind to their antigens by non-covalent interactions; they use hydrogen bonds, Van der Waals forces or electrostatic forces. Depending on the structure of the antibody, which varies with isotype, and the structure of the antigen, an antibody may have either one (monovalent) binding interaction with an antigen, or multiple simultaneous (multivalent) interactions.[1]
The strength of the binding interaction between a single Fab region of an antibody and a single antigenic epitope is called the affinity of the antibody. When a single antigen has more than one epitope for the same antibody, more than one interaction may occur between the antigen and antibody; the antigen in this instance is known as a multivalent or polyvalent antigen. Antibodies can bind each epitope of a polyvalent antigen with each Fab region they possess. In other words, monomeric antibodies like IgG that have two Fab regions (one at each tip of the Y) can simultaneously bind to two epitopes, while pentameric antibodies like IgM that have ten Fab regions can simulataneously bind up to ten epitopes. Each Fab region houses an identical variable domain and will have the same affinity for the epitope, but the cumulation of multiple, simultaneous antibody–antigen interactions will create an overall stronger binding of the antigen. This compound affinity of multiple antibody–antigen interactions with a single antigen is known as the antibody avidity. Avidity can be orders of magnitude greater than affinity helping, for instance, poorly affinity-matured but highly multivalent IgM still bind antigen efficiently.[26]
Since antibodies exist freely in the bloodstream or bound to cell membranes, they are said to be part of the humoral immune system. The circulating antibodies are produced by clonal B cells that are specific to only one antigen, a virus hull protein fragment, for example. In binding their specific antigens, the antibodies can cause agglutination and precipitation of antibody-antigen products primed for phagocytosis by macrophages and other cells, block viral receptors, and stimulate other immune responses, such as the complement pathway.[27]
Antibodies that recognize viruses can block these by binding them directly.[3] In doing so, the virus is unable to dock to its preferred receptor for infecting a host cell. Some antibodies, like IgA, also directly bind to microbes in mucus to prevent the colonization of mucosal tissues. Antibodies, like those in antivenoms, neutralize toxins by binding to them.[28] Certain viruses may be able to evade the immune system if antibody neutralization is inadequate. For example, when viruses such as HIV, are not completely covered by neutralizing antibody, the presence of the antibodies can enhance viral infectivity instead of inhibiting it; HIV prefers to infect the cells that bind to antibodies.[29] Antibodies cannot attack pathogens within cells, and certain viruses (such as HIV, HSV and HBV) "hide" inside cells for long periods of time to avoid them.[3] 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 reactivate later, causing a resurgence of symptoms.[3]
Antibodies are clonally generated for binding single specific antigens. Antibodies can link these viruses or cells together, causing them to agglutinate, or coagulate, so that phagocytes can capture them more effectively.[3]
Antibodies that bind to surface antigens on, for example a bacterium, attract the first component of the complement cascade with their Fc region and initiate activation of the "classical" complement system.[27] This results in the killing of bacteria in two ways.[3] First, the binding of the antibody and complement molecules marks the microbe for ingestion by phagocytes in a process called opsonization. These phagocytes are attracted by some of the complement molecules that are generated in the complement cascade. Secondly, some complement system components form a membrane attack complex to assist the antibodies killing the bacteria directly.[30]
Mast cells and phagocytes have specific receptors on their cell surface that bind antibodies. These are called Fc receptors, and, as the name suggests, these receptors interact with the Fc region of IgA, IgG, and IgE antibodies. The engagement of a particular antibody with the Fc receptor on a particular cell triggers the effector function of that cell (e.g. phagocytes will phagocytose, mast cells will degranulate) that will ultimately result in destruction of the invading microbe. The Fc receptors are isotype-specific, which gives a great flexibility to the immune system, because different situations require only certain immune mechanisms to respond to antigens.[1]
Detection of particular antibodies is a very common form of medical diagnostics, and applications such as serology depend on these methods.[31] In biochemical assays for disease diagnosis,[32] a titer of Epstein-Barr virus or Lyme disease detects antibodies produced by the body that are specific to those antigens in the blood. If those antibodies are not present, either the person has not been infected, or the infection occurred a very long time ago, and the antibodies have naturally decayed.
Autoimmune disorders can often be traced to antibodies that bind the body's own epitopes; many can be detected through blood tests. Antibodies directed against red blood cell surface antigens in immune mediated hemolytic anemia are detected with the Coombs test.[33] The Coombs test is also used for antibody screening in blood transfusion preparation and also for antibody screening in antenatal women.[33]
"Targeted" monoclonal antibody therapy is employed to treat diseases such as rheumatoid arthritis,[34] multiple sclerosis,[35] psoriasis,[36] and in many forms of cancer including non-Hodgkin's lymphoma,[37] colorectal cancer, head and neck cancer and breast cancer.[38]
Some immune deficiencies, such as X-linked agammaglobulinemia and hypogammaglobulinemia, result in partial or complete lack of antibodies.[39] These diseases are often treated by inducing a short term form of immunity called passive immunity. Passive immunity is achieved through the transfer of ready made antibodies in the form of human or animal serum, pooled immunoglobulin or monoclonal antibodies, into the affected individual.[40]
Elevations in different classes of immunoglobulins are sometimes useful in determining the cause of liver damage in patients whom the diagnosis is unclear.[2] For example, elevated IgA indicates alcoholic cirrhosis, elevated IgM indicates viral hepatitis and primary biliary cirrhosis, while IgG is elevated in viral hepatitis, autoimmune hepatitis and cirrhosis.
RHOGAM antibodies are a trade name for Rho(D) Immune Globulin antibodies specific to the human Rh D antigen.[41] The Rhesus factor (also known as the D antigen) is an antigen found on red blood cells in the blood. People that are Rh+ have this antigen on their red blood cells. People that are Rh- don't have this antigen on their red blood cells.
In the course of regular childbirth, delivery trauma or other prenatal complications can cause blood from the fetus to occasionally enter the mother's system. In the case of an Rh-incompatible mother and child, blood mixing from this trauma may 'sensitize' the Rh-negative mother to the Rh antigen, putting the remainder of the pregnancy, and any subsequent pregnancies, at risk for hemolytic disease of the newborn.[42] RhoGAM is administered as part of a pre-natal treatment regimen to prevent any sensitization that may occur when a Rhesus-negative mother has a fetus that is Rhesus-positive.
Treatment of an mother with RhoGAM antibodies prior to and immediately after trauma and delivery destroys any Rh antigen incidentally in the mother's system from the fetus. Importantly, this will happen before the antigen can stimulate the mother's memory-mediated immune response B cells to "remember" Rh antigen. Therefore, her humoral immune system will never be stimulated to make anti-Rh antibodies, and will not attack the current, or any potential subsequent, baby's Rhesus antigens. RhoGAM prevents 'sensitization' that can lead to Rh disease, but does not prevent or treat the underlying disease itself.[41]
Specific antibodies are often produced by injecting the antigen into a small mammal, such as a mouse or rabbit and purified for use in research. Sometimes, in order to obtain large quantity of antibodies, goats, sheep, or horses are used. Blood isolated from these animals contains polyclonal antibodies -- multiple antibodies that bind to the same antigen. The serum, also known as the antiserum, now contains the desired antibodies, that are often purified with Protein A/G purification or antigen affinity chromatography.[43] To obtain a antibody that is specific for a single epitope of an antigen, the lymphocytes that produce the antibodies are isolated and immortalized by fusing them with a cancer cell line. The fused cells are called hybridomas, and will continually grow and secrete antibody in culture. Single hybridoma cells are isolated, during this procedure, to generate cell clones that all produce the same antibody; these antibodies are called monoclonal antibodies.
In research, purified antibodies are used in many applications. They are most commonly used to identify and locate intracellular and extracellular proteins. Antibodies are used in flow cytometry to differentiate cell types by the combinations of proteins they express.[44] They are also used in immunoprecipitation to separate proteins (and anything bound to them; co-immunoprecipitation) from the other molecules in a cell lysate,[45] in Western blot analyses to identify proteins separated by electrophoresis,[46] in immunohistochemistry to examine protein expression in tissue sections,[47] and to examine the localization of proteins within cells by immunofluorescence.[44] Proteins can also be detected and quantified with antibodies, using ELISA and ELISPOT techniques.[48][49]
<gallery> Image:Antibody scheme.svg|Antibody scheme Image:Anticorps.png|Antibody Image:Immunoglobulin basic unit.svg|Immunoglobulin basic unit Image:Scfv1.png|A Y-shaped antibody molecule. </gallery>
Contents