An antibody ( Ab), also known as an immunoglobulin ( Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and Viral disease. The antibody recognizes a unique molecule of the pathogen, called an antigen. Each tip of the "Y" of an antibody contains a paratope (analogous to a lock) that is specific for one particular epitope (analogous to a key) on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a microbe or an infected cell for attack by other parts of the immune system, or can neutralize it directly (for example, by blocking a part of a virus that is essential for its invasion).
To allow the immune system to recognize millions of different antigens, the antigen-binding sites at both tips of the antibody come in an equally wide variety. In contrast, the remainder of the antibody is relatively constant. It only occurs in a few variants, which define the antibody's class or isotype: IgA, IgD, IgE, IgG, or IgM. The constant region at the trunk of the antibody includes sites involved in interactions with other components of the immune system. The class hence determines the function triggered by an antibody after binding to an antigen, in addition to some structural features. Antibodies from different classes also differ in where they are released in the body and at what stage of an immune response.
Together with B and , antibodies are the most important part of the adaptive immune system.
They occur in two forms: attached to a B cell or in soluble form in extracellular fluids such as blood plasma.
Initially, antibodies are attached to the surface of a B cell – they are then referred to as (BCR).
After an antigen binds to a BCR, the B cell activates to proliferate and differentiate into either , which secrete soluble antibodies with the same paratope, or memory B cells, which survive in the body to enable long-lasting immunity to the antigen.
Soluble antibodies are released into the blood and tissue fluids, as well as many .
Because these fluids were traditionally known as humorism, antibody-mediated immunity is sometimes known as, or considered a part of, humoral immunity.
Antibodies are belonging to the immunoglobulin superfamily. The terms antibody and immunoglobulin are often used interchangeably, though the term 'antibody' is sometimes reserved for the secreted, soluble form, i.e. excluding B-cell receptors.
In humans and most , an antibody unit consists of four polypeptide chains; two identical heavy chains and two identical light chains connected by .
Each chain is a series of protein domain: somewhat similar sequences of about 110 each.
These domains are usually represented in simplified schematics as rectangles.
Light chains consist of one variable domain VL and one constant domain CL, while heavy chains contain one variable domain VH and three to four constant domains CH1, CH2, …
Structurally an antibody is also partitioned into two antigen-binding fragments (Fab), containing one VL, VH, CL, and CH1 domain each, as well as the crystallisable fragment (Fc), forming the trunk of the Y shape.
In between them is a hinge region of the heavy chains, whose flexibility allows antibodies to bind to pairs of epitopes at various distances, to form complexes (protein dimer, trimers, etc.), and to bind effector molecules more easily.
In an electrophoresis test of blood proteins, antibodies mostly migrate to the last, gamma globulin fraction.
Conversely, most gamma-globulins are antibodies, which is why the two terms were historically used as synonyms, as were the symbols Ig and gamma.
This variant terminology fell out of use due to the correspondence being inexact and due to confusion with γ heavy chains which characterize the IgG class of antibodies.
The existence of two identical antibody-binding sites allows antibody molecules to bind strongly to multivalent antigen (repeating sites such as in bacterial cell walls, or other sites at some distance apart), as well as to form antibody complexes and larger antigen-antibody complexes. The resulting cross-linking plays a role in activating other parts of the immune system.
The structures of CDRs have been clustered and classified by Chothia et al.
and more recently by North et al.
and Nikoloudis et al.
In the framework of the immune network theory, CDRs are also called idiotypes. According to immune network theory, the adaptive immune system is regulated by interactions between idiotypes.
Another role of the Fc region is to selectively distribute different antibody classes across the body. In particular, the neonatal Fc receptor (FcRn) binds to the Fc region of IgG antibodies to transport it across the placenta, from the mother to the fetus.
Antibodies are , that is, they have carbohydrates (glycans) added to conserved amino acid residues.
These conserved glycosylation sites occur in the Fc region and influence interactions with effector molecules.
Antibodies also form complexes by binding to antigen: this is called an antigen-antibody complex or immune complex.
Small antigens can cross-link two antibodies, also leading to the formation of antibody dimers, trimers, tetramers, etc.
Multivalent antigens (cells with multiple epitopes) can form larger complexes with antibodies.
An extreme example is the clumping, or agglutination, of red blood cells with antibodies in the Coombs test to determine : the large clumps become insoluble, leading to visually apparent precipitatation.
The classes differ in their biological properties, functional locations and ability to deal with different antigens, as depicted in the table.
For example, IgE antibodies are responsible for an allergic response consisting of histamine release from , contributing to asthma. The antibody's variable region binds to allergic antigen, for example house dust mite particles, while its Fc region (in the ε heavy chains) binds to Fc receptor ε on a mast cell, triggering its degranulation: the release of molecules stored in its granules.
The antibody isotype of a B cell changes during cell development and activation. Immature B cells, which have never been exposed to an antigen, express only the IgM isotype in a cell surface bound form. The B lymphocyte, in this ready-to-respond form, is known as a "naive B lymphocyte." The naive B lymphocyte expresses both surface IgM and IgD. The co-expression of both of these immunoglobulin isotypes renders the B cell ready to respond to antigen.
Antibody and antigen interact by spatial complementarity (lock and key). The molecular forces involved in the Fab-epitope interaction are weak and non-specific – for example electrostatic forces, , hydrophobic interactions, and van der Waals forces. This means binding between antibody and antigen is reversible, and the antibody's affinity towards an antigen is relative rather than absolute. Relatively weak binding also means it is possible for an antibody to Cross-reactivity with different antigens of different relative affinities.
More indirectly, an antibody can signal immune cells to present antibody fragments to , or downregulate other immune cells to avoid autoimmunity.
Activated B cells differentiate into either antibody-producing cells called that secrete soluble antibody or memory cells that survive in the body for years afterward in order to allow the immune system to remember an antigen and respond faster upon future exposures.
At the prenatal and neonatal stages of life, the presence of antibodies is provided by passive immunization from the mother. Early endogenous antibody production varies for different kinds of antibodies, and usually appear within the first years of life. Since antibodies exist freely in the bloodstream, they are said to be part of the humoral immune system. Circulating antibodies are produced by clonal B cells that specifically respond to only one antigen (an example is a virus capsid fragment). Antibodies contribute to immunity in three ways: They prevent pathogens from entering or damaging cells by binding to them; they stimulate removal of pathogens by macrophages and other cells by coating the pathogen; and they trigger destruction of pathogens by stimulating other such as the complement pathway. Antibodies will also trigger vasoactive amine degranulation to contribute to immunity against certain types of antigens (helminths, allergens).
Those cells that recognize coated pathogens have Fc receptors, which, as the name suggests, 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 an effector function of that cell; phagocytes will phagocytosis, and will degranulation, natural killer cells will release and cytotoxic molecules; that will ultimately result in destruction of the invading microbe. The activation of natural killer cells by antibodies initiates a cytotoxic mechanism known as antibody-dependent cell-mediated cytotoxicity (ADCC) – this process may explain the efficacy of monoclonal antibodies used in biological therapies against cancer. The Fc receptors are isotype-specific, which gives greater flexibility to the immune system, invoking only the appropriate immune mechanisms for distinct pathogens.
RAG proteins play an important role with V(D)J recombination in cutting DNA at a particular region. Without the presence of these proteins, V(D)J recombination would not occur.
After a B cell produces a functional immunoglobulin gene during V(D)J recombination, it cannot express any other variable region (a process known as allelic exclusion) thus each B cell can produce antibodies containing only one kind of variable chain.
This serves to increase the diversity of the antibody pool and impacts the antibody's antigen-binding affinity. Some point mutations will result in the production of antibodies that have a weaker interaction (low affinity) with their antigen than the original antibody, and some mutations will generate antibodies with a stronger interaction (high affinity). B cells that express high affinity antibodies on their surface will receive a strong survival signal during interactions with other cells, whereas those with low affinity antibodies will not, and will die by apoptosis. Thus, B cells expressing antibodies with a higher affinity for the antigen will outcompete those with weaker affinities for function and survival allowing the average affinity of antibodies to increase over time. The process of generating antibodies with increased binding affinities is called affinity maturation. Affinity maturation occurs in mature B cells after V(D)J recombination, and is dependent on help from helper T cells.
Class switching occurs in the heavy chain gene locus by a mechanism called class switch recombination (CSR). This mechanism relies on conserved nucleotide motifs, called switch (S) regions, found in DNA upstream of each constant region gene (except in the δ-chain). The DNA strand is broken by the activity of a series of at two selected S-regions. The variable domain exon is rejoined through a process called non-homologous end joining (NHEJ) to the desired constant region (γ, α or ε). This process results in an immunoglobulin gene that encodes an antibody of a different isotype.
To further improve the function of heterodimeric antibodies, many scientists are looking towards artificial constructs. Artificial antibodies are largely diverse protein motifs that use the functional strategy of the antibody molecule, but aren't limited by the loop and framework structural constraints of the natural antibody. Being able to control the combinational design of the sequence and three-dimensional space could transcend the natural design and allow for the attachment of different combinations of drugs to the arms.
Heterodimeric antibodies have a greater range in shapes they can take and the drugs that are attached to the arms don't have to be the same on each arm, allowing for different combinations of drugs to be used in cancer treatment. Pharmaceuticals are able to produce highly functional bispecific, and even multispecific, antibodies. The degree to which they can function is impressive given that such a change of shape from the natural form should lead to decreased functionality.
The study of antibodies began in 1890 when Emil von Behring and Kitasato Shibasaburō described antibody activity against diphtheria and . Von Behring and Kitasato put forward the theory of
Further work concentrated on characterizing the structures of the antibody proteins. A major advance in these structural studies was the discovery in the early 1960s by Gerald Edelman and Joseph Gally of the antibody light chain, and their realization that this protein is the same as the Bence-Jones protein described in 1845 by Henry Bence Jones. Edelman went on to discover that antibodies are composed of disulfide bond-linked heavy and light chains. Around the same time, antibody-binding (Fab) and antibody tail (Fc) regions of Immunoglobulin G were characterized by Rodney Porter. Together, these scientists deduced the structure and complete amino acid sequence of IgG, a feat for which they were jointly awarded the 1972 Nobel Prize in Physiology or Medicine. The Fv fragment was prepared and characterized by David Givol. While most of these early studies focused on IgM and IgG, other immunoglobulin isotypes were identified in the 1960s: Thomas Tomasi discovered secretory antibody (IgA); David S. Rowe and John L. Fahey discovered IgD; and Kimishige Ishizaka and Teruko Ishizaka discovered IgE and showed it was a class of antibodies involved in allergic reactions. In a landmark series of experiments beginning in 1976, Susumu Tonegawa showed that genetic material can rearrange itself to form the vast array of available antibodies.
In clinical immunology, levels of individual classes of immunoglobulins are measured by nephelometry (or turbidimetry) to characterize the antibody profile of patient. Elevations in different classes of immunoglobulins are sometimes useful in determining the cause of liver damage in patients for whom the diagnosis is unclear. 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.
Autoimmune disorders can often be traced to antibodies that bind the body's own ; many can be detected through . Antibodies directed against red blood cell surface antigens in immune mediated hemolytic anemia are detected with the Coombs test. The Coombs test is also used for antibody screening in blood transfusion preparation and also for antibody screening in antenatal women.
Practically, several immunodiagnostic methods based on detection of complex antigen-antibody are used to diagnose infectious diseases, for example ELISA, immunofluorescence, Western blot, immunodiffusion, immunoelectrophoresis, and magnetic immunoassay. Antibodies raised against human chorionic gonadotropin are used in over the counter pregnancy tests.
New dioxaborolane chemistry enables radioactive fluoride (18F) labeling of antibodies, which allows for positron emission tomography (PET) imaging of cancer.
Some immune deficiencies, such as X-linked agammaglobulinemia and hypogammaglobulinemia, result in partial or complete lack of antibodies. 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 blood plasma, pooled immunoglobulin or monoclonal antibodies, into the affected individual.
Rho(D) immune globulin antibodies are specific for human RhD antigen. Anti-RhD antibodies are administered as part of a prenatal care to prevent sensitization that may occur when a Rh-negative mother has a Rh-positive fetus. Treatment of a mother with Anti-RhD antibodies prior to and immediately after trauma and delivery destroys Rh antigen in the mother's system from the fetus. It is important to note that this occurs before the antigen can stimulate maternal B cells to "remember" Rh antigen by generating memory B cells. Therefore, her humoral immune system will not make anti-Rh antibodies, and will not attack the Rh antigens of the current or subsequent babies. Rho(D) Immune Globulin treatment prevents sensitization that can lead to Rh disease, but does not prevent or treat the underlying disease itself.
In research, purified antibodies are used in many applications. Antibodies for research applications can be found directly from antibody suppliers, or through use of a specialist search engine. Research antibodies are most commonly used to identify and locate intracellular and extracellular proteins. Antibodies are used in flow cytometry to differentiate cell types by the proteins they express; different types of cell express different combinations of cluster of differentiation molecules on their surface, and produce different intracellular and secretable proteins. They are also used in immunoprecipitation to separate proteins and anything bound to them (co-immunoprecipitation) from other molecules in a cell lysate, in Western blot analyses to identify proteins separated by electrophoresis, and in immunohistochemistry or immunofluorescence to examine protein expression in tissue sections or to locate proteins within cells with the assistance of a microscope.
Antibodies used in research are some of the most powerful, yet most problematic reagents with a tremendous number of factors that must be controlled in any experiment including cross reactivity, or the antibody recognizing multiple epitopes and affinity, which can vary widely depending on experimental conditions such as pH, solvent, state of tissue etc. Multiple attempts have been made to improve both the way that researchers validate antibodies and ways in which they report on antibodies. Researchers using antibodies in their work need to record them correctly in order to allow their research to be reproducible (and therefore tested, and qualified by other researchers). Less than half of research antibodies referenced in academic papers can be easily identified. Papers published in F1000 in 2014 and 2015 provide researchers with a guide for reporting research antibody use. The RRID paper, is co-published in 4 journals that implemented the RRIDs Standard for research resource citation, which draws data from the antibodyregistry.org as the source of antibody identifiers (see also group at Force11).
The ability to describe the antibody through binding affinity to the antigen is supplemented by information on antibody structure and amino acid sequences for the purpose of patent claims. Several methods have been presented for computational design of antibodies based on the structural bioinformatics studies of antibody CDRs.
There are a variety of methods used to sequence an antibody including Edman degradation, cDNA, etc.; albeit one of the most common modern uses for peptide/protein identification is liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). High volume antibody sequencing methods require computational approaches for the data analysis, including de novo sequencing directly from tandem mass spectra and database search methods that use existing protein sequence databases. Many versions of shotgun protein sequencing are able to increase the coverage by utilizing CID/HCD/ETD fragmentation methods and other techniques, and they have achieved substantial progress in attempt to fully sequence proteins, especially antibodies. Other methods have assumed the existence of similar proteins, a known genome sequence, or combined top-down and bottom up approaches. Current technologies have the ability to assemble protein sequences with high accuracy by integrating de novo sequencing peptides, intensity, and positional confidence scores from database and homology searches.
Antigen-binding site
Fc region
Protein structure
Antibody complexes
B cell receptors
Classes
+ Antibody isotypes of mammals
! Class Description Found in mucosal areas, such as the gut, respiratory tract and urogenital tract, and prevents colonization by . Also found in saliva, tears, and breast milk. Functions mainly as an antigen receptor on B cells that have not been exposed to antigens. It has been shown to activate and to produce antimicrobial factors. Binds to and triggers histamine release from and , and is involved in allergy. Also protects against . In its four forms, provides the majority of antibody-based immunity against invading pathogens. The only antibody capable of crossing the placenta to give passive immunity to the fetus. Expressed on the surface of B cells (monomer) and in a secreted form (pentamer) with very high avidity. Eliminates pathogens in the early stages of B cell-mediated (humoral) immunity before there is sufficient IgG.
Light chain types
In animals
+ Antibody classes not found in mammals Found in and ; related to mammalian IgG. Found in Elasmobranchii; related to mammalian IgD.
Antibody–antigen interactions
Function
Activation of complement
Activation of effector cells
Natural antibodies
Immunoglobulin diversity
Domain variability
V(D)J recombination
Somatic hypermutation and affinity maturation
Class switching
Specificity designations
Asymmetrical antibodies
History
/ref> His idea prompted Paul Ehrlich to propose the side-chain theory for antibody and antigen interaction in 1897, when he hypothesized that receptors (described as "side-chains") on the surface of cells could bind specifically to – in a "lock-and-key" interaction – and that this binding reaction is the trigger for the production of antibodies. Other researchers believed that antibodies existed freely in the blood and, in 1904, Almroth Wright suggested that soluble antibodies coated bacteria to label them for phagocytosis and killing; a process that he named .
In the 1920s, Michael Heidelberger and Oswald Avery observed that antigens could be precipitated by antibodies and went on to show that antibodies are made of protein. The biochemical properties of antigen-antibody-binding interactions were examined in more detail in the late 1930s by John Marrack. The next major advance was in the 1940s, when Linus Pauling confirmed the lock-and-key theory proposed by Ehrlich by showing that the interactions between antibodies and antigens depend more on their shape than their chemical composition. In 1948, Astrid Fagraeus discovered that B cell, in the form of , were responsible for generating antibodies.
Medical applications
Disease diagnosis
Disease therapy
Prenatal therapy
Research applications
Regulations
Production and testing
Before clinical trials
Preclinical studies
Structure prediction and computational antibody design
WAM and Prediction of Immunoglobulin Structure (PIGS)
Prediction of Immunoglobulin Structure (PIGS) enables computational modeling of antibody variable regions. Rosetta Antibody is a novel antibody FV region structure prediction server, which incorporates sophisticated techniques to minimize CDR loops and optimize the relative orientation of the light and heavy chains, as well as homology models that predict successful docking of antibodies with their unique antigen.
RosettaAntibody
Antibody mimetic
See also
External links
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