Information AboutAntibodies |
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Antibodies are Y-shaped 1(1): e16. Although the general structure of all antibodies is very similar, a small region at the tip of the protein is extremely variable, allowing millions of antibodies with slightly different tip structures to exist. Each of these variants can bind to a different target, known as an Antigen .1 This huge diversity of antibodies allows the immune system to recognize an equally wide diversity of antigens. The unique part of the antigen recognized by an antibody is called an Epitope . These epitopes fit precisely with their antibody, similar to a key fitting into a lock, in a highly specific interaction that allows antibodies to identify and bind only their unique antigen in the midst of the millions of different molecules that make up an Organism . Recognition of an antigen by an antibody ''tags'' it for attack by other parts of the immune system. Antibodies can also neutralize targets directly by, for example, binding to a part of a Pathogen that it needs to cause an infection.2 The large and diverse population of antibodies is generated by random combinations of a set of Gene segments that encode different antigen binding sites (or ''paratopes''), followed by random Mutation s in this area of the antibody gene, which create further diversity.3 Antibody genes also re-organize in a process called Class Switching that changes the base of the heavy chain to another, creating a different isotype of the antibody that retains the antigen specific variable region. This allows a single antibody to be used by several different parts of the immune system. Antibodies occur in two forms: a Soluble form Secreted into the blood and Tissue fluids, and a Membrane-bound form attached to the surface of a B Cell that is called the ''B cell receptor'' (BCR). The BCR allows a B cell to detect when a specific antigen is present in the body and triggers B cell activation.4 Activated B cells Differentiate into either antibody generating factories called Plasma Cell s that secrete soluble antibody, or into Memory Cell s that survive in the body for years afterwards to allow the immune system to remember an antigen and respond faster upon future exposures.5 Antibodies are, therefore, an essential component of the Adaptive Immune System that learns, adapts and remembers responses to invading pathogens. Production of antibodies is the main function of the Humoral Immune System .6 ISOTYPES Antibodies can come in different forms known as Isotypes or classes. In mammals there are five antibody isotypes known as IgA, IgD, IgE,IgG and IgM. They are each named with an "Ig" prefix that stands for immunoglobulin, another name for antibody, and differ in their biological properties, functional locations and ability to deal with different antigens, as depicted in the table.9 The antibody isotype of a B cell changes during the cell's Development and Activation . Immature B cells, which have never been exposed to antigen, are known as naïve B cells and express only the IgM isotype in a cell surface bound form. B cells begin to express both IgM and IgD when they reach maturity - the co-expression of both these immunoglobulin isotypes renders the B cell 'mature' and ready to respond to antigen.10 B cell activation follows engagement of the cell bound antibody 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 antibody in a Secrete d form rather than a Membrane -bound form. Some Daughter Cell s of the activated B cells undergo Isotype Switching , a mechanism that causes the production of antiodies to change from IgM or IgD to the other antibody isotypes, IgE, IgA or IgG, that have defined roles in the immune system. STRUCTURE Antibodies are heavy Globular Plasma Protein s that are also known as immunoglobulins. They have sugar chains added to some of their Amino Acid residues.11 In other words, antibodies are '' Glycoprotein s''. The basic functional unit of each antibody is an immunoglobulin (Ig) Monomer (containing only one Ig unit); secreted antibodies can also be Dimer ic with two Ig units as with IgA, Tetramer ic with four Ig units like Teleost Fish IgM, or Pentamer ic with five Ig units, like mammalian IgM.12 Immunoglobulin domains 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 Bond s. Each chain is composed of Structural Domain s called Ig domains. These domains contain about 70-110 Amino Acid s 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 Sheet s create a “sandwich” shape, held together by interactions between conserved Cysteine s and other charged amino acids. Heavy chain There are five types of mammalian Ig s. 2. Fc Region 3. Heavy Chain with one variable (VH) domain followed by a constant domain (CH1), a hinge region, and two more constant (CH2 and CH3) domains. 4. Light Chain with one variable (VL) and one constant (CL) domain 5. Antigen binding site (paratope) 6. Hinge regions]] Each heavy chain has two regions, the ''constant region'' and the ''variable region''. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains γ, α and δ have a constant region composed of ''three'' tandem (in a line) Ig Domains , and a hinge region for added flexibility; heavy chains μ and ε have a constant region composed of ''four'' immunoglobulin domains. The variable region of the heavy chain differs in antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or B Cell Clone . The variable region of each heavy chain is approximately 110 amino acids long and is composed of a single Ig domain. Light chain In mammals there are only two types of s like Chondrichthyes and Teleostei . Fab and Fc Regions Some parts of an antibody have unique functions. The tip of the Y, for example, contains the site that binds antigen and, therefore, recognizes specific foreign objects. This region of the antibody is called the '' Fab (fragment, Antigen Binding) Region ''. It is composed of one constant and one variable domain from each heavy and light chain of the antibody.14 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 plays a role in modulating immune cell activity. 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. By binding to specific proteins the Fc region ensures that each antibody generates an appropriate immune response for a given antigen.15 The Fc region also binds to various cell Receptor s, such as Fc Receptor s, 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 .16 FUNCTION Since antibodies exist freely in the bloodstream, they are said to be part of the and other cells by coating the pathogen; and they can trigger direct pathogen destruction by stimulating other Immune Response s such as the Complement Pathway .17 Neutralization Viruses and intracellular bacteria must enter a cell to begin replication; they gain entry into the cell by binding to specific molecules on the cell surface. Antibodies that recognize viruses block the ability of the pathogen to dock to its preferred receptor by binding them directly. Bound virus is unable to infect a host cell. Some antibodies, like IgA, directly bind to microbes in Mucus to prevent colonization of Mucosal Tissue s, and those in Antivenoms neutralize Toxin s by binding to them.18 Some viruses are able to evade the immune system when antibody neutralization is inadequate. For example, when viruses such as HIV , are not completely covered by neutralizing antibody, the antibodies may Enhance viral Infectivity instead of inhibiting it; HIV prefers to infect cells that bind to antibodies.19 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 neutralization. During Chronic diseases such as Herpes , an outbreak is quickly suppressed by the immune system, but some cells retain virus that will reactivate later and cause a resurgence of symptoms; the infection is never truly Eradicate d. Activation of complement 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. This results in the killing of bacteria in two ways. 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 certain complement molecules generated in the complement cascade. Secondly, some complement system components form a Membrane Attack Complex to assist antibodies to kill the bacterium directly.20 Activation of effector cells To combat pathogens that replicate outside cells antibodies bind to pathogens to link them together, causing them to Agglutinate . Since an antibody possesses at least two paratopes it can bind more than one antigen by binding identical epitopes carried on the surfaces of these antigens. By coating the pathogen, antibodies stimulate effector functions against the pathogen in cells that recognize their Fc region. Those cells which recognize coated pathogens have Fc receptors which, as the name suggests, interacts 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 Phagocytose , Mast Cell s and Neutrophil s will Degranulate , Natural Killer Cell s will release Cytokine s and Cytotoxic molecules; that will ultimately result in destruction of the invading microbe. The Fc receptors are isotype-specific, which gives greater flexibility to the immune system, invoking only the appropriate immune mechanisms for distinct pathogens. has five Ig units. Each Ig unit (labeled 1) has two epitope binding Fab Region s, so IgM is capable of binding up to 10 epitopes.]] AFFINITY VERSUS AVIDITY Antibodies reversibly bind to their antigens by Non-covalent interactions; they use Hydrogen Bond s, Van Der Waals Force s or Electrostatic Force s. Depending on the structure of the antibody and the structure of the antigen, an antibody may have either one ( Monovalent ) binding interaction with an antigen, or multiple simultaneous ( Multivalent ) interactions. The strength of the binding interaction between a single paratope of an antibody and a single antigenic epitope is termed the '' Affinity '' of the antibody. When an 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 simultaneously bind up to ten epitopes. The cumulative effect of multiple, simultaneous antibody–antigen interactions is an increase in the affinity of the antibody for the antigen. The total affinity of an antibody with more than one binding site is defined as its '' Avidity ''. Avidity can be orders of magnitude greater than affinity helping, for instance, highly multivalent IgM bind antigen efficiently despite its poor affinity.21 Antibody affinity and avidity play an important role in protecting against infection, since the higher the affinity of an antibody for its antigen the lower the amount of antibody required to eliminate the antigen. This is because antibodies with higher affinity will bind to, and eliminate an antigen when lower concentrations of the antigen are present in the body. Antibodies with lower affinity or avidity require more antigen (and thus pathogen) be present, when it will be more difficult to control the spread of the pathogen. 22 IMMUNOGLOBULIN DIVERSITY Virtually all microbes can trigger an antibody response. Successful recognition and eradication of many different types of microbes requires diversity among antibodies; their amino acid composition varies allowing them to interact with many different antigens.23 It has been estimated that humans generate about 10 billion different antibodies, each capable of binding a distinct epitope of an antigen.24 Although a huge repertoire of different antibodies is generated in a single individual, the number of Gene s available to make these proteins is limited. Several complex genetic mechanisms have evolved that allow vertebrate B cells to generate a diverse pool of antibodies from a relatively small number of antibody genes.25 V(D)J recombination , and thus different antigen specificities. 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.26 Somatic hypermutation and affinity maturation For more details on this topic, see Somatic Hypermutation and Affinity Maturation Another mechanism that generates antibody diversity occurs in the mature B cell. Following activation with antigen, B cells begin to Proliferate rapidly. In these rapidly dividing cells, the genes encoding the variable domains of the heavy and light chains undergo a high rate of Point Mutation , by a process called ''somatic hypermutation'' (SHM). SHM results in approximately one Nucleotide change per variable gene, per cell division. As a consequence, any daughter B cells will acquire slight Amino Acid differences in the variable domains of their antibody chains. Somatic hypermutation serves to increase the diversity of the antibody pool and impacts the antibody’s antigen-binding Affinity .27 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).28 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 higher affinity antibodies for will outcompete those with weaker affinities for function and survival. 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 Cell s.29 Class switching Isotype Or Class Switching is a Biological Process occurring after activation of the B cell, which allows the cell to produce different classes of antibody (IgA, IgE, or IgG). The different classes of antibody, and thus effector functions, are defined by the constant (C) regions of the immunoglobulin heavy chain. Initially, naïve B cells express only cell-surface IgM and IgD with identical antigen binding regions. Each isotype is adapted for a distinct function, therefore, after activation, an antibody with a IgG, IgA, or IgE effector function might be required to effectively eliminate an antigen. Class switching allows different daughter cells from the same activated B cell to produce antibodies of different isotypes. Only the constant region of the antibody heavy chain changes during class switching; the variable regions, and therefore antigen specificity, remain unchanged. Thus the progeny of a single B cell can produce antibodies, all specific for the same antigen, but with the ability to produce the effector function appropriate for each antigenic challenge. Class switching is triggered by cytokines; the isotype generated depends on which cytokines are present in the B cell environment.30 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 Enzyme s at two selected S-regions.3132 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.33 MEDICAL APPLICATIONS Disease diagnosis Detection of particular antibodies is a very common form of medical Diagnostics , and applications such as Serology depend on these methods.34 For example, in biochemical assays for disease diagnosis,35 a Titer of antibodies directed against Epstein-Barr Virus or Lyme Disease is estimated from the blood. If those antibodies are not present, either the person is not infected, or the infection occurred a ''very'' long time ago, and the B cells generating these specific antibodies have naturally decayed. In Clinical Immunology , levels of individual classes of immunoglobulins are measured by Nephelometry (or turbidimetry) to characterize the antibody profile of patient.36 Elevations in different classes of immunoglobulins are sometimes useful in determining the cause of Liver damage in patients 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 Disorder s can often be traced to antibodies that bind the body's own Epitope s; many can be detected through Blood Test s. Antibodies directed against Red Blood Cell surface antigens in immune mediated Hemolytic Anemia are detected with the Coombs Test .37 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 , and Immunoelectrophoresis . Disease therapy "Targeted" Monoclonal Antibody therapy is employed to treat diseases such as Rheumatoid Arthritis ,38 Multiple Sclerosis ,39 Psoriasis ,40 and many forms of Cancer including Non-Hodgkin's Lymphoma ,41 Colorectal Cancer , Head And Neck Cancer and Breast Cancer .42 Some immune deficiencies, such as X-linked Agammaglobulinemia and Hypogammaglobulinemia , result in partial or complete lack of antibodies.43 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.44 Prenatal therapy ''Rho(D) Immune Globulin'' antibodies are specific for human Rhesus D antigen, also known as Rhesus Factor .45 These antibodies are known under several Brand Names , including RhoGAM. Rhesus factor is an Antigen found on Red Blood Cell s; individuals that are Rhesus-positive (Rh+) have this antigen on their red blood cells and individuals that are Rhesus-negative (Rh-) do not. During normal Childbirth , delivery trauma or complications during pregnancy, blood from a Fetus can enter the mother's system. In the case of an Rh-incompatible mother and child, consequential blood mixing may sensitize an Rh- mother to the Rh antigen, putting the remainder of the Pregnancy , and any subsequent pregnancies, at risk for Hemolytic Disease Of The Newborn .46 RhoGAM is administered as part of a Prenatal Treatment Regimen to prevent sensitization that may occur when a Rhesus-negative mother has a Rhesus-positive fetus. Treatment of a mother with RhoGAM antibodies prior to and immediately after trauma and delivery destroys Rh antigen in the mother's system from the fetus. Importantly, this occurs before the antigen can stimulate maternal B cells to "remember" Rh antigen by generating Memory B Cell s. Therefore, her Humoral Immune System will not make anti-Rh antibodies, and will not attack the Rhesus antigens of the current or subsequent baby. RhoGAM treatment prevents sensitization that can lead to Rh Disease , but does not prevent or treat the underlying disease itself. RESEARCH APPLICATIONS image of the eukaryotic Cytoskeleton . Actin filaments are shown in red, Microtubule s in green, and the Nuclei In Blue .]] Specific antibodies are produced by injecting an antigen into a Mammal , such as a Mouse , Rat or Rabbit for small quantities of antibody, or Goat , Sheep , or Horse for large quantities of antibody. Blood isolated from these animals contains '' Polyclonal Antibodies '' — multiple antibodies that bind to the same antigen — in the Serum , which can now be called Antiserum . Antigens are also injected into Chicken s for generation of polyclonal antibodies in Egg Yolk .47 To obtain antibody that is specific for a single epitope of an antigen, antibody-secreting Lymphocyte s are isolated from the animal and Immortalized by fusing them with a cancer cell line. The fused cells are called Hybridoma s, and will continually grow and secrete antibody in culture. Single hybridoma cells are isolated by Dilution Cloning to generate Cell Clones that all produce the same antibody; these antibodies are called '' Monoclonal Antibodies ''.48 Generated polyclonal and monoclonal antibodies are often purified using Protein A/G or Antigen-affinity Chromatography .49 Use 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 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.50 They are also used in Immunoprecipitation to separate proteins and anything bound to them (co-immunoprecipitation) from other molecules in a Cell Lysate ,51 in Western Blot analyses to identify proteins separated by Electrophoresis ,52 and in Immunohistochemistry or Immunofluorescence to examine protein expression in tissue sections or to locate proteins within cells with the assistance of a Microscope .53 Proteins can also be detected and quantified with antibodies, using ELISA and ELISPOT techniques.5455 HISTORY See Also: History of immunology The study of antibodies began in 1890 when Emil Von Behring and Shibasaburo Kitasato described antibody activity against Diphtheria and Tetanus Toxin s. Behring and Kitasato put forward the theory of Humoral Immunity , proposing that a mediator in Serum could react with a foreign antigen.5657 Their 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 Toxin s – in a "lock-and-key" interaction – and that this binding reaction was the trigger for the production of antibodies.58 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 Opsonin ization.59 In the 1920s, Michael Heidelberger and Oswald Avery observed that antigens could be precipitated by antibodies and went on to show that antibodies were made of protein.60 The biochemical properties of antigen-antibody binding interactions were examined in more detail in the late 1930s by John Marrack .61 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 depended more on their shape than their chemical composition.62 In 1948, Astrid Fagreaus discovered that B cells, in the form of Plasma Cell s, were responsible for generating antibodies.63 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 discovered secretory antibody ( IgA ) 67 and David Rowe and John Fahey identified IgD,68 and IgE was identified by Kikishige Ishizaka and Teruki Ishizaka as a class of antibodies involved in allergic reactions.69 Genetic studies revealed the basis of the vast diversity of these antibody proteins when somatic recombination of immunoglobulin genes was identified by Susumu Tonegawa in 1976.70 SEE ALSO REFERENCES EXTERNAL LINKS |
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